Tifundvo letehlukene tekuvikela titjalo tiniketa sicondziso lesibalulekile setifundvo sekuchaza emaphethini ekugaya lokukhetsekile kwetitjalo, kepha kucagela kwabo lokubalulekile kusatawuhlolwa. Lapha, sasebentisa kuhlatiya kwe-tandem mass spectrometry (MS/MS) lokungakhetsi kuhlola ngendlela lehlelekile i-metabolome yetinhlobo letincishiswe ligwayi kusuka etitjalweni ngamunye kuya etincenyeni tebantfu kanye netinhlobo letihlobene kakhulu, futsi sacubungula linani lelikhulu lemibono yesici se-mass spectrometric lesekelwe ku-spectra lehlanganisiwe kulolwati. Luhlaka lwekuhlola kucagela lokubalulekile kwekuvikela lokukahle (OD) kanye netifundvo letihlosiwe letihambako (MT). Lengcenye yelwatiso lwe metabolomics yetitjalo ihambisana ne OD theory, kepha iphikisana nekubiketela lokuyinhloko kwe MT theory ngekugucuka kwe metabolomics lokubangelwa tilwane letidla titjalo. Kusuka ku micro kuya ku macro esikalini sekutigucukela kwemvelo, luphawu lwe jasmonate lwakhonjwa njengentfo lebalulekile ye OD, kantsi luphawu lwe ethylene lwaniketa kulungiswa lokuhle kwemphendvulo lekhetsekile ye herbivore lebhalwe yi MS/MS molecule network.
Ema metabolites lakhetsekile lanetakhiwo letehlukahlukene ngiwo lahlanganyela kakhulu ekuvumelaniseni kwetitjalo nemvelo, ikakhulukati ekuvikeleni titsa (1). Kwehlukahlukana lokumangalisako kwekugaya kudla lokukhetsekile lokutfolakala etitjalweni kukhutsate emashumi eminyaka elucwaningo lolujulile ngetincenye tayo letinyenti temisebenti yemvelo, futsi kwakha luhlu loludze lwetifundvo tekuvikela titjalo, lokungukutfutfuka kwekutigucukela kwemvelo kanye nekwemvelo kwekusebentisana kwetitjalo netinambuzane. Lucwaningo lolufakazelako luniketa sicondziso lesibalulekile (2). Kepha, letifundvo tekuvikela titjalo atizange tilandzele indlela lejwayelekile yekucabanga lokukhiphako lokucabangako, lapho khona kucagela lokubalulekile bekusesigabeni lesifanako sekuhlatiya (3) futsi kwahlolwa ngekulinga kwentela kuchubela embili umjikeleto lolandzelako wekutfutfukiswa kwetifundvo (4). Imikhawulo yetebuchwepheshe ivimbela kuhlanganiswa kwemininingwane etigabeni letitsite tekugaya kudla futsi ayifaki kuhlatiya lokuphelele kwe-metabolites lekhetsekile, ngaleyo ndlela kuvimbela kucatsanisa lokusemkhatsini wetigaba lokubalulekile ekuthuthukiseni theoretical (5). Kushoda kwemininingwane lephelele ye metabolomics kanye nemali levamile yekucatsanisa kuhamba kwemsebenti wekucubungula sikhala sekugaya emkhatsini wemacembu etitjalo letehlukene kuvimbela kuvutfwa kwesayensi yalensimu.
Intfutfuko yakamuva emkhakheni we tandem mass spectrometry (MS/MS) metabolomics ingaveta ngalokuphelele tingucuko tekugaya ngekhatsi nasemkhatsini wetinhlobo te clade yeluhlelo loluniketiwe, futsi ingahlanganiswa netindlela tekubala kubala kufana kwesakhiwo emkhatsini waletingcucuko letiyinkimbinkimbi. Lwati lwangaphambilini lwekhemikhali (5). Kuhlanganiswa kwetebucwepheshe lobutfutfukile ekuhlaziyeni nasekusebentiseni emakhompyutha kuniketa luhlaka loludzingekako lwekuhlolwa kwesikhatsi lesidze kwekubiketela lokuningi lokwentiwa ngetifundvo temvelo kanye nekutigucukela kwemvelo kwekuhlukahlukana kwemetabolic. Shannon (6) wetfula i-information theory kwekucala ngca esihlokweni sakhe se-seminal nga-1948, abeka sisekelo sekuhlatiya lwati ngetibalo, lolusetjentiswe emikhakheni leminyenti ngaphandle kwekusetjentiswa kwalo kwekucala. Ku genomics, lwati theory isetjentiswe ngemphumelelo kulinganisa kulandzelana lwati conservative (7). Kulucwaningo lwe-transcriptomics, ithiyori yelwati ihlatiya tingucuko jikelele ku-transcriptome (8). Kulucwaningo lwangaphambilini, sasebentisa luhlaka lwetibalo lwe-theory yelwati ku-metabolomics kuchaza bungcweti be-metabolic belizinga leticubu etitjalweni (9). Lapha, sihlanganisa MS/MS-based workflow kanye neluhlaka lwetibalo lwe theory yelwati, lolubonakala ngekuhlukahluka kwe metabolic emalini levamile, kucatsanisa kucagela lokubalulekile kwe theory yekuvikela titjalo ye metabolome lekhutsatwa tilwane letidla titjalo.
Letinhlaka tetifundvo tekuvikela titjalo tivame kufaka ekhatsi futsi tingahlukaniswa tibe tigaba letimbili: leto letizama kuchaza kusabalalisa kwema-metabolites lakhetsekile esitjalweni ngekuya ngemisebenti yekuvikela, njengekuvikela lokukahle (OD) (10), lisu lelihambako (MT) (11) ) Futsi kubukeka kabusha ngesikhatsi (12) theory of others in, . kutfolakala kutsintsa kukhula kwetitjalo kanye nekugcwala kwema metabolites lakhetsekile, njenge carbon: umcabango wekulinganisa umsoco (13), umcabango welizinga lekukhula (14 ), kanye nemcabango wekulingana kwekukhula kanye nekuhlukana (15). Lamasethi lamabili etifundvo asemazingeni lehlukene ekuhlatiya (4). Nomakunjalo, tifundvo letimbili letifaka ekhatsi imisebenti yekuvikela esigabeni sekusebenta tibusa inkhulumo mayelana nekuvikela kwetitjalo letisungulako kanye nekuvikela lokungentiwa: i-OD theory, letsatsa kutsi titjalo titjala imali etivikeleni tato letibitako temakhemikhali kuphela uma kudzingeka, sibonelo, uma tihlaselwa Uma silwane setjani sihlasela, ngako-ke, ngekuya kwe- umsebenti uyabelwa (10); le MT hypothesis iphakamisa kutsi kute i-axis yekushintja kwe metabolite lecondzile, kepha le metabolite iyashintja ngekungakhetsi, ngaleyondlela kudala kungenteka Kuvimbela “lihloso lekunyakata” lekuhlasela tilwane letidla titjalo. Ngamanye emagama, letifundvo letimbili tenta kucagela lokuphikisako mayelana nekuvuselelwa kwemetabolic lokwenteka ngemuva kwekuhlaselwa kwetilwane letidla titjalo: budlelwane emkhatsini wekuhlanganiswa kwe-unidirectional kwema-metabolites ngemsebenti wekuvikela (OD) kanye netingucuko te-metabolic letingacondziswa (MT) (11 ).
Le OD kanye ne MT hypotheses ayifaki kuphela tingucuko letibangelwe ku metabolome, kepha nemiphumela yemvelo kanye nekutigucukela kwemvelo kwekugcwala kwalama metabolites, njengetindleko tekuvumelana nesimo kanye netinzuzo taletingucuko te metabolic esimeni lesitsite semvelo (16). Noma ngabe totimbili letinkhulumo tibona umsebenti wekuvikela wema metabolites lakhetsekile, langabiza noma angabiti, kubiketela lokubalulekile lokwehlukanisa letinkhulumo te OD ne MT kulele ekucondziseni kwetingucuko te metabolic letibangelwako. Kubiketela kwe-OD theory kutfole kunakwa kakhulu kwekuhlola kuze kube manje. Letivivinyo tifaka ekhatsi kufundza imisebenti yekuvikela lecondzile noma lengacondzile yeticubu letehlukene tetinhlanganisela letitsite ema greenhouses kanye netimo temvelo, kanye netingucuko esigabeni sekutfutfuka kwetitjalo (17-19). Kepha, kuze kube ngunyalo, ngenca yekungabikhona kwekuhamba kwemsebenti kanye neluhlaka lwetibalo lwekuhlatiya lokuphelele kwemhlaba wonkhe kwekuhlukahlukana kwekugaya kudla kwanoma ngusiphi silwane, umehluko lomkhulu wekubiketela emkhatsini waletifundvo letimbili (lokusho kutsi, sicondziso setingucuko tekugaya kudla) kusafanele kutsi kuhlolwe. Lapha, siniketa kuhlatiya lokunjalo.
Lokunye lokubaluleke kakhulu kwema metabolites lakhetsekile etitjalweni kuhlukahluka kwawo lokukhulu kwesakhiwo kuwo onkhe emazinga kusuka etitjalweni letingatodvwa, bantfu kuya etilwaneni letifanako (20). Tingucuko letiningi tesilinganiso kuma metabolites lakhetsekile tingabonakala esikalini sebantfu, kantsi umehluko locinile wekhwalithi uvame kugcinwa esigabeni setinhlobo (20). Ngakoke, kwehlukahlukana kwe metabolic yetitjalo kuyintfo lebalulekile yekuhlukahlukana lokusebenta, lokukhombisa kuvumelana nema niches lehlukene, ikakhulu lawo ma niches lanematfuba lehlukene ekuhlasela ngetinambuzane letikhetsekile kanye netilwane letidla titjalo letivamile (21). Kusukela endzabeni ya Fraenkel (22) lehamba embili ngetizatfu tekubakhona kwema metabolites lakhetsekile etitjalweni, kusebentisana netinambuzane letehlukene bekutsatfwa njengekucindzeteleka lokubalulekile kwekukhetsa, futsi lokusebentisana kukholelwa kutsi kwakha titjalo ngesikhatsi sekutigucukela kwemvelo. Indlela yekugaya kudla (23). Umehluko emkhatsini wetinhlobo ngekwehlukahlukana kwema metabolites lakhetsekile kungaphindze kukhombise ibhalansi yemtimba lehambisana nekuvikela kwetitjalo letisungulako naletingavumeleki kumasu ekudla titjalo, njengoba letinhlobo letimbili tivame kuhambisana kabi nalomunye nalomunye (24). Noma kungaba yinzuzo kugcina kuvikela lokuhle ngasosonkhe sikhatsi, tingucuko te metabolic ngesikhatsi letihambisana nekuvikela tiniketa tinzuzo leticacile ekuvumeleni titjalo kutsi tibeke tinsita letibalulekile kuletinye tintfo tekutjala imali temtimba (19, 24), kanye nekugwema sidzingo se symbiosis. Umonakalo wesibambiso (25). Ngekwengeta, lokuhlelwa kabusha kwema metabolites lakhetsekile labangelwa tilwane letidla titjalo letinambuzane kungaholela ekusakatekeni lokubhubhisako kubantfu (26), futsi kungakhombisa kufundvwa lokucondzile kwetingucuko temvelo letinkhulu ku jasmonic acid (JA) signal, lekungagcinwa kubantfu. Timphawu te JA letisetulu naletiphansi titsengisa emkhatsini wekuvikela tilwane letidla titjalo kanye nekuncintisana netinhlobo letitsite (27). Ngekwengeta, tindlela letikhetsekile te-metabolite biosynthetic titawulahlekelwa ngekushesha kanye nekugucuka ngesikhatsi sekutigucukela kwemvelo, lokuholela ekusabalalisweni kwe-metabolic le-patchy emkhatsini wetinhlobo letihlobene kakhulu (28). Leti polymorphisms tingasungulwa ngekushesha ngekuphendvula kushintja kwemaphethini etilwane letidla titjalo (29), lokusho kutsi kugucugucuka kwemimango yetilwane letidla titjalo kuyintfo lebalulekile lecondzisa i-metabolic heterogeneity.
Lapha, sasombulula ngalokukhetsekile letinkinga letilandzelako. (I) Lesinambuzane lesidla titjalo siphindze silungise njani i-metabolome yesitjalo? (Ii) Ngutiphi tincenye letiyinhloko telwatiso te-metabolic plasticity letingalinganiswa kute kuhlolwe kucagela kwe-theory yekuvikela sikhatsi lesidze? (Iii) Noma ngabe kuhlelwe kabusha i-metabolome yesitjalo ngendlela lehlukile kuloyo lohlaselako, uma kunjalo, nguyiphi indzima ledlalwa yi-hormone yesitjalo ekwenteni imphendvulo letsite yekugaya kudla, futsi ngutiphi ti-metabolites letifaka sandla ekukhetseni kwetinhlobo tekuvikela? (Iv) Njengoba kucagela lokwentiwa ngetifundvo letiningi tekuvikela tinganwetshwa kuwo onkhe emazinga eticubu tetilwane, sabuta kutsi imphendvulo yekugaya kudla lebangelwako ihambisana kangakanani kusuka ekucatsaniselweni kwangekhatsi kuya ekucatsaniselweni kwetinhlobo letihlukene? Kute kwentiwe loku, sifundze ngendlela lehlelekile i-metabolome yemacembe e-nicotine yeligwayi, lokusitjalo sesibonelo semvelo lesinekugaya lokukhetsekile lokunothile, futsi iyasebenta kahle kulwa netibungu tetilwane letimbili letidla titjalo tendzabuko, iLepidoptera Datura (Ms) ( Lenelulaka kakhulu, idliwa kakhulu) Ku-Solanaceae littorala ye-SpodoS “genus”, netitjalo letibambako te Solanaceae kanye naletinye tibambako taletinye tihlobo kanye nemindeni Kudla kwetitjalo. Sihlatiye i-MS / MS metabolomics spectrum futsi sakhipha lwati lwetifundvo tetibalo tekuchaza kucatsanisa tifundvo te-OD ne-MT. Dala emabalave lakhetsekile kute uveze bunjalo bema-metabolites labalulekile. Lokuhlatiya kwandluliswa kuya kubantfu bendzabuko be N. nasi kanye netinhlobo teligwayi letihlobene kakhulu kwentela kuhlatiya kabanti i-covariance emkhatsini wekukhombisa emahomoni esitjalo kanye nekufakwa kwe OD.
Kute sitsatse imephu jikelele mayelana ne-plasticity kanye nesakhiwo se-metabolome yemacembe egwayi ledla titjalo, sasebentisa kuhlatiya lokutfutfukiswe ngaphambilini kanye nekuhamba kwemsebenti wekubala kute sihlanganise ngalokuphelele futsi si-deconvolute idatha lesetulu ye-resolution letimele ye-MS / MS spectra kusuka etitjalweni letikhishwako ( 9). Lendlela lengahlukaniswa (lebitwa ngekutsi yi MS/MS) ingakha ema spectra lahlanganisiwe langadzingeki, langabese asetjentiswa kukho konkhe kuhlatiya kwelizinga lehlanganisiwe lokuchazwe lapha. Letintfo letigucukako tetitjalo letihlanganisiwe titinhlobo letehlukene, letifaka emakhulu kuya etinkhulungwaneni tetitjalo letigucukako (cishe 500-1000-s/MS/MS lapha). Lapha, sicabanga ngepulasitiki yekugaya kudla eluhlaka lwethiyori yelwati, futsi silinganise kwehlukahlukana kanye nebungcweti be-metabolome lesekelwe ku-Shannon entropy yekusabalalisa imvamisa yekugaya kudla. Kusebentisa ifomula lesetjentiswe ngaphambilini (8), sabala isethi yetinkhomba letingasetjentiswa kulinganisa kwehlukahlukana kwe-metabolome (inkomba ye-Hj), kukhetseka kwephrofayli ye-metabolic (inkomba ye-δj) kanye nekukhetseka kwe-metabolic ye-metabolite yinye (inkomba ye-Si) . Ngetulu kwaloko, sasebentisa Inkomba Yepulasitiki Yelibanga Lelihlobene (RDPI) kulinganisa kusebenta kwe-metabolome kwetilwane letidla titjalo (Umfanekiso 1A) (30). Ngaphakatsi kwaloluhlaka lwetibalo, siphatsa i-MS / MS spectrum njengeyunithi yelwati lolusisekelo, bese sicubungula buningi lobuhlobene be-MS / MS ibe yimephu yekusakateka kwemvamisa, bese sisebentisa i-Shannon entropy kulinganisa kwehlukahlukana kwe-metabolome kusuka kuyo. Le metabolome lekhetsekile ilinganiswa ngesilinganiso sekucaciswa kwe-MS / MS spectrum yinye. Ngakoke, kukhula kwebuningi balamanye emakilasi eMS/MS ngemuva kwekufakwa kwetilwane letidla titjalo kuguculwa kube yi spectral inducibility, iRDPI kanye nekukhetseka, lokusho kutsi, kukhula kwe δj index, ngoba ema metabolites lakhetsekile kakhulu ayakhicitwa futsi kukhicitwa inkomba lesetulu ye Si. Kwehliswa kwenkomba yekuhlukahlukana kwe Hj kukhombisa kutsi noma linani le MS/MS leyakhiwe liyancishiswa, noma kusabalalisa imvamisa yephrofayli kuyashintja ngendlela lengafani, ngesikhatsi kuncishiswa kungaciniseki kwayo jikelele. Ngekubalwa kwe Si index, kungenteka kutsi kugcizelelwe kutsi nguyiphi iMS/MS lekhutsatwa tilwane letitsite letidla titjalo, ngekuphikisana, nguyiphi iMS/MS lengaphendvuli ku induction, lokuyinkhomba lebalulekile yekwehlukanisa MT kanye ne OD kucagela.
(A) Tichazamagama tetibalo letisetjentiselwa tilwane letidla titjalo (H1 kuya ku Hx) MS / MS idatha-inducibility (RDPI), kwehlukahlukana (Hj inkomba), kukhetseka (δj inkomba) kanye ne-metabolite lekhetsekile (Si inkomba). Kukhula kwelizinga lekukhetseka (δj) kukhombisa kutsi, ngesilinganiso, ema metabolites lakhetsekile ladla titjalo atawukhicitwa, kantsi kwehla kwekuhlukahluka (Hj) kukhombisa kwehla kwekukhicita ema metabolites noma kusabalalisa lokungalingani kwema metabolites kumephu yekusakateka . Linani le Si lihlola kutsi ngabe le metabolite icacile esimeni lesiniketwe sona (lapha, herbivorous) noma ngekuphikisa igcinwe esigabeni lesifanako. (B) Umdvwebo wemcondvo wekubiketela kwethiyori yekuvikela kusetjentiswa i-axis yethiyori yelwati. OD theory ibiketela kutsi kuhlaselwa kwetilwane letidla titjalo kutawukhulisa ema metabolites ekuvikela, ngaleyondlela kukhulise δj. Ngesikhatsi lesifanako, Hj uyancipha ngoba iphrofayli ihlelwa kabusha kuya ekunciphiseni kungaciniseki kwelwati lwe-metabolic. Le MT theory ibiketela kutsi kuhlaselwa kwetilwane letidla titjalo kutawubangela tingucuko letingacondzi ku metabolome, ngaleyondlela kukhulise iHj njengenkhomba yekukhuphuka kwekungaciniseki kwelwati lwe metabolic futsi kubangele kusabalalisa lokungahleliwe kwe Si. Siphindze saphakamisa imodeli lehlanganisiwe, i-MT lencono kakhulu, lapho khona lamanye ema-metabolites lanemanani laphakeme ekuvikela atawukhuliswa kakhulu (linani leliphakeme le-Si), kantsi lamanye akhombisa timphendvulo letingahleliwe (linani leliphansi le-Si).
Kusebentisa tinchazelo tethiyori yelwati, sihumusha ithiyori ye-OD kucagela kutsi tingucuko te-metabolite lekhetsekile letibangwa tilwane letidla titjalo esimeni se-metabolite lesingakabangelwa titawuholela (i) ekwandziseni kwekukhetseka kwe-metabolic (Si index) lokushayela kukhetseka kwe-metabonomic (δj index) Kukhula kwe) emacembu latsite e-metabolite lakhetsekile e-de metaboloij lene-defense lephakeme ( i-metaboloiH) index) ngenca yekushintja kwekusakateka kwemvamisa ye-metabolic kuya ekusakateni kwemtimba we-leptin kakhulu. Esigabeni se metabolite yinye, kusabalalisa kwe Si lokuyalwe kulindzelekile, lapho khona le metabolite itawukhulisa linani le Si ngekuya kwelinani layo lekuvikela (Umfanekiso 1B). Kulomugca, sichaza i-MT theory kucagela kutsi kujabula kutawuholela (i) tingucuko letingacondzi kuma-metabolites letiholela ekuncipheni kwe-δj index, kanye (ii) nekwandziswa kwe-Hj index ngenca yekwandziswa kwekungaciniseki kwe-metabolic. Noma kungahleleki, lokungalinganiswa yi Shannon entropy ngendlela yekuhlukahluka lokujwayelekile. Njengobe kunjalo ngekwakheka kwe-metabolic, i-MT theory itawubiketela kusabalalisa lokungahleliwe kwe-Si. Kucabangela kutsi ema-metabolites latsite angaphansi kwetimo letitsite ngaphansi kwetimo letitsite, futsi letinye timo atikho ngaphansi kwetimo letitsite, futsi linani labo lekuvikela lincike endzaweni, saphindze saphakamisa imodeli yekuvikela lehlanganisiwe, lapho δj ne-Hj isabalaliswa khona kabili eceleni kwe-Si Kukhula kuto tonkhe ticondziso letitsite metabolite lesetulu emacembu atawukhulisa, leyo Si babe inani le-metabolite lelitsite lelingahleliwe. kusabalalisa (Umfanekiso 1B).
Kute sivivinye kucagela kwemfundziso yekuvikela lechazwe kabusha ku-axis yenchazelo yemfundziso yelwati, sakhulisa tibungu letidla titjalo letinguchwepheshe (Ms) noma jikelele (Sl) emacembe eNepenthes pallens (Umfanekiso 2A). Kusebentisa kuhlatiya kwe MS/MS, satfola 599 MS/MS spectra lengadzingeki (ifayela lemininingwane S1) kusuka ku methanol extracts yeticubu temacembe letibutfwe ngemuva kwekudla kwemacimbi. Kusebentisa RDPI, Hj, kanye δj indexes kubona ngemehlo engcondvo reconfiguration lokucuketfwe lwati kumafayela MS / MS ukuhleleka kuveta emaphethini latsakatelisako (Umfanekiso 2B). Lokuhamba embili jikelele kutsi, njengoba kuchazwe ngumchazi welwati, njengoba emacimbi achubeka nekudla emacembe, lizinga lakho konkhe kuhlelwa kabusha kwe metabolic liyakhula ngekuhamba kwesikhatsi: emahora langemashumi lasikhombisa nakubili ngemuva kwekudla kwe herbivore, iRDPI ikhula kakhulu. Nawucatsanisa nekulawula lokungalimali, iHj yancishiswa kakhulu, lebekubangelwa lizinga lelikhulile lekukhetsekile kwephrofayli yekugaya, lebeyilinganiswa yi-δj index. Lomkhuba lobonakalako uyahambisana nekubiketela kwe OD theory, kepha akuhambisani nekubiketela lokuyinhloko kwe MT theory, lekholelwa kutsi tingucuko letingahleliwe (letingacondzi) emazingeni e metabolite tisetjentiswa njenge camouflage yekuvikela (Umfanekiso 1B). Noma nje lokucuketfwe kwe oral secretion (OS) elicitor kanye nekutiphatsa kwekudla kwaletilwane letimbili letidla titjalo kwehlukile, kudla kwato lokucondzile kwaholela ekugucukeni lokufanako eticondzisweni te Hj na δj ngesikhatsi sekuvuna semahora lamashumi lamabili nakune na 72. Umehluko kuphela wenteka emahoreni langu-72 e-RDPI. Nawucatsanisa naloko lokubangelwa kudla kwa Ms, umjikeleto jikelele lobangwa kudla kwe Sl bewuphakeme.
(A) Kuklanywa kwekuhlola: ingulube levamile (S1) noma tilwane letidla titjalo letinguchwepheshe (Ms) tidliwa ngemacembe lakhiwe luswayi etitjalweni te pitcher, kantsi ngekudla titjalo letilingisiwe, iOS ya Ms (W + OSMs) isetjentiselwa kuphatsa kubhobota kwetikhundla temacembe letisemgangatfweni tesilonda. S1 (W + OSSl) tibungu noma emanti (W + W). Lokulawula (C) licembe lelingakalimali. (B) Inducibility (RDPI kucatsaniswa neshadi lekulawula), kwehlukahlukana (Hj inkomba) kanye nekukhetsekile (δj inkomba) inkomba lebalwe imephu lekhetsekile ye-metabolite (599 MS / MS; ifayela lemininingwane S1). Tinkhanyeti tikhombisa umehluko lomkhulu emkhatsini wekudla lokucondzile kwetilwane letidla titjalo kanye nelicembu lekulawula (T-test yemfundzi nge t-test lebhanjiwe, *P<0.05 kanye na ***P<0.001). ns, akubalulekile. (C) Inkomba yekusombulula sikhatsi ye main (libhokisi leliluhlata sasibhakabhaka, i-amino acid, i-esidi yetilwane kanye nashukela; ifayela lemininingwane S2) kanye ne-spectrum lekhetsekile ye-metabolite (libhokisi lelibovu 443 MS/MS; lifayela lemininingwane S1) ngemuva kwekwelashwa kwekudla kwetitjalo lokulingisiwe. Ibhande umbala libhekisela 95% sikhatsi sekwetsemba. Inkhanyeti ikhombisa umehluko lomkhulu emkhatsini wekwelashwa nekulawula [kuhlatiya kwe-quadratic kwekuhluka (ANOVA), kulandzelwe umehluko lomkhulu ngekwetsembeka waTukey (HSD) wekucatsanisa lokunyenti kwe-post hoc, * P <0.05, ** P <0.01 Futsi *** P <0.001]. (D) Kukhetseka kwetindzawo tekusakateka kanye nemaphrofayli lakhetsekile e-metabolite (emasampula laphindzaphindziwe ngetindlela tekwelapha letehlukene).
Kuhlola kutsi ngabe kuvuselelwa lokubangwa tilwane letidla titjalo esigabeni se metabolome kubonakala yini etingucukweni letisesigabeni setilwane letidla titjalo ngamunye, sacala sagcila kuma metabolites lafundvwa ngaphambilini emacembe eNepenthes pallens ngekumelana kwetilwane letidla titjalo letifakazelwe. Ema amides e Phenolic ngema conjugates e hydroxycinnamamide-polyamine labutsana ngesikhatsi senchubo yekudla titjalo tetinambuzane futsi tatiwa kutsi tinciphisa kusebenta kwetinambuzane (32). Siseshe tintfo letihamba embili te-MS / MS lehambisanako futsi sakhetsa emagoba abo lahlanganisiwe e-kinetic (Umfanekiso S1). Akumangalisi, tintfo letisuselwa ku phenol letingahlanganyeli ngalokucondzile ekuvikeleni tilwane letidla titjalo, njenge chlorogenic acid (CGA) kanye ne rutin, tilawulwa phansi ngemuva kwekudla titjalo. Ngalokwehlukile, tilwane letidla titjalo tingenta ema phenol amides abe nemandla lamakhulu. Kudla lokuchubekako kwaletilwane letimbili letidla titjalo kwaholela ekucishe kube nekujabula lokufanako kwema phenolamides, futsi lendlela beyibonakala kakhulu ngekuhlanganiswa kwe de novo kwema phenolamides. Lesimanga lesifanako sitawubonakala uma kuhlolwa indlela ye-17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), lekhicita linani lelikhulu le-diterpenes le-acyclic lenemisebenti lephumelelako yekulwa netilwane letidla titjalo (33), lapho khona Ms.
Lokungakalungi lokungenteka kwekuhlolwa kwekudla kwetilwane letidla titjalo ngalokucondzile ngumehluko esigabeni sekudla kwemacembe kanye nesikhatsi sekudla kwetilwane letidla titjalo, lokwenta kube matima kucedza imiphumela lekhetsekile yetilwane letidla titjalo lebangelwa tilondza kanye netilwane letidla titjalo. Kute silungise kancono tinhlobo tetilwane letidla titjalo letikhetsekile tekuphendvula kwekugaya emacembe lokubangelwako, salingisa kudla kwetibungu te Ms kanye ne Sl ngekutsi sisheshe sifake iOS lesanda kubutfwa (OSM kanye ne OSS1) ku puncture lejwayelekile W yetindzawo temacembe letingagucuki. Lenchubo ibitwa ngekutsi yi W + OS kwelashwa, futsi ilinganisa kungeniswa ngekutsi ibeke sikhatsi lesifanele sekucala kwemphendvulo lekhicitwa yi herbivore ngaphandle kwekubangela imiphumela ledidako yemehluko esigabeni noma linani lekulahlekelwa kweticubu (Umfanekiso 2A) (34). Kusebentisa kuhlatiya kwe MS/MS kanye nephayiphi yekubala, satfola 443 MS/MS spectra (ifayela lemininingwane S1), leyahlangana ne spectra lebeyihlanganiswe ngaphambilini kusuka ekuhlolweni kwekudla lokucondzile. Kuhlatiya kwemfundziso yelwati lwalesethi yedatha ye MS/MS kwakhombisa kutsi kuhlelwa kabusha kwema metabolomes lakhetsekile emacembe ngekulingisa tilwane letidla titjalo kwakhombisa kukhutsata lokukhetsekile kwe OS (Umfanekiso 2C). Ikakhulukati, uma kucatsaniswa nekwelashwa kwe-OSS1, i-OSM yabangela kutfutfukiswa kwekukhetsekile kwe-metabolome emahoreni langu-4. Kufanele kutsi kucashelwe kutsi uma kucatsaniswa nesethi yedatha yekuhlola yekudla kwetilwane letidla titjalo leticondzile, i-metabolic kinetics lebonakala esikhaleni lesinetigaba letimbili isebentisa iHj ne δj njengetikhonjiso kanye nekucondziswa kwekukhetsekile kwe metabolome ngekuphendvula kwelashwa kwetilwane letidla titjalo letilingisiwe ngekuhamba kwesikhatsi Kukhula lokuhambisanako (Umfanekiso 2D). Ngesikhatsi lesifanako, salinganisa lokucuketfwe yi-amino acid, i-organic acid kanye nashukela (ifayela lemininingwane S2) kuphenya kutsi ngabe loku kwandza lokuhlosiwe kwebungcweti be-metabolome kungenca yekuhleleka kabusha kwe-carbon metabolism lesemkhatsini ngekuphendvula tilwane letidla titjalo letilingisiwe ( Umfanekiso S2). Kuchaza kancono lephethini, sachubeka sagadza i-kinetics yekugcwala kwe-metabolic ye-phenolamide lebekukhulunywe ngayo ngaphambilini kanye netindlela te-17-HGL-DTG. Lokufakwa kwe OS-lokukhetsekile kwetilwane letidla titjalo kuguculwa kube yindlela yekuhlela kabusha leyehlukile ngaphakatsi kwekugaya kwe phenolamide (Umfanekiso S3). Ema amides ePhenolic lacuketse tincenye te coumarin kanye ne caffeoyl akhetsa kukhutsatwa yi OSS1, kantsi ema OSM avusa kungeniswa lokutsite kwema conjugates eferulyl. Kulendlela ye-17-HGL-DTG, kufaka umehluko we-OS ngemikhicito ye-malonylation kanye ne-dimalonylation lesezansi kwatfolakala (Umfanekiso S3).
Ngemuva kwaloko, safundza ngepulasitiki ye-transcriptome lebangelwa yi-OS sisebentisa isethi yedatha ye-microarray yesikhatsi, lelingisa kusetjentiswa kwema-OSM kwelapha emacembe emacembe esitjalo se-rosette etilwaneni letidla titjalo. I-kinetics yekutsatsa sampula ngekuyinhloko ihlangana ne-kinetics lesetjentiswe kulolu cwaningo lwe-metabolomics (35). Nawucatsanisa ne-metabolome reconfiguration lapho khona i-plasticity ye-metabolic ikhuphuka kakhulu ngekuhamba kwesikhatsi, sibona kuqhuma kwekubhala kwesikhashana emacembe lakhutsatwa ngu-Ms, lapho khona i-transcriptome inducibility (RDPI) kanye nekukhetseka (δj) kukhona ku-1 Kwaba nekwandzisa lokukhulu emahoreni, kanye nekuhlukahluka lokubalulekile (Hj) lokuncishiswe kakhulu ngu-BMPx bekungu-BMP1. yekukhetseka kwe-transcriptome (Umfanekiso S4). Imindeni yetifo tekugaya (njenge P450, glycosyltransferase, kanye ne BAHD acyltransferase) ihlanganyela enchubeni yekuhlanganisa emafutsa lakhetsekile kusuka kumayunithi esakhiwo lasuselwa ekugayeni lokuyinhloko, kulandzelwa imodeli leshiwo ngenhla yekucala lesetulu-lekhetsekile. Njengesifundvo sendzaba, indlela ye-phenylalanine yahlaziywa. Lokuhlatiya kucinisekisile kutsi emajini layinhloko ku phenolamide metabolism akhutsatwa kakhulu yi OS etilwaneni letidla titjalo uma kucatsaniswa netitjalo letingakhangwa, futsi ahambisana kakhulu ngendlela yato yekuveta. Sici sekubhala MYB8 kanye nemajini esakhiwo PAL1, PAL2, C4H kanye 4CL etulu kwalendlela kwakhombisa kucala kusenesikhatsi kwekubhala. Ema Acyltransferases ladlala indzima emhlanganweni wekugcina we phenolamide, njenge AT1, DH29, kanye ne CV86, akhombisa iphethini yekulawula sikhatsi lesidze (Umfanekiso S4). Loku lokubonwe ngenhla kukhombisa kutsi kucala kwekucala kwekukhetsekile kwe-transcriptome kanye nekutfutfukiswa kwekucala kwe-metabolomics kamuva kuyindlela lehlanganisiwe, lekungaba ngenca yeluhlelo lwekulawula loluhambisanako lolucala imphendvulo lenemandla yekuvikela.
Lokuhleleka kabusha ekuboniseni emahomoni etitjalo kusebenta njengelucwencwe lolulawulako loluhlanganisa lwati lwetilwane letidla titjalo kwentela kuhlela kabusha iphysiology yetitjalo. Ngemuva kwekulingisa kwetilwane letidla titjalo, sakala emandla lahlanganisiwe etigaba temahomoni etitjalo letibalulekile futsi sabona ngemehlo engcondvo kuveta ngekubambisana kwesikhatsi emkhatsini wato [Pearson correlation coefficient (PCC)> 0.4] (Umfanekiso 3A). Njengoba kulindzelekile, emahomoni esitjalo lahlobene ne biosynthesis ahlanganiswa ngaphakatsi kwenethiwekhi yekuveta emahomoni esitjalo ngekubambisana. Ngekwengeta, kucaciswa kwe metabolic (Si index) kukhonjiswa kulenethiwekhi kwentela kugcizelela emahomoni esitjalo labangelwa kwelashwa lokuhlukene. Tindzawo letimbili letiyinhloko tekuphendvula lokutsite kwe herbivorous tiyadvwetjwa: lenye isesigabeni se JA, lapho khona iJA (indlela yayo lesebentako yetilwane iJA-Ile) kanye naletinye tintfo letisuselwa ku JA tikhombisa emaphuzu laphakeme eSi; lomunye ngu-ethileni (ET). iGibberellin yakhombisa kuphela kukhula lokulinganiselwe kwekukhetsa kwetilwane letidla titjalo, kantsi lamanye emahomoni etitjalo, njenge cytokinin, auxin, kanye ne abscisic acid, bekanekukhetsa lokuphansi kwekufaka tilwane letidla titjalo. Nawucatsanisa nekusebentisa W + W kuphela, kukhuliswa kwelinani leliphakeme le-JA derivatives ngekusebentisa sicelo se-OS (W + OS) kungaguculwa ngalokuyinhloko kube yinkomba lecinile lekhetsekile ye-JAs. Ngekungalindzeleki, i-OSM ne-OSS1 lenetintfo letehlukene te-elicitor kwatiwa kutsi kubangela kugcwala lokufanako kwe-JA ne-JA-Ile. Ngalokwehlukile ku-OSS1, i-OSM ikhutsatwa ngalokukhetsekile futsi ngamandla ngema-OSM, kantsi i-OSS1 ayikhulisi imphendvulo yetilonda te-basal (Umfanekiso 3B).
(A) Kuhlatiya inethiwekhi yekuveta ngekubambisana lokusekelwe ekubaleni kwe-PCC kwekulingisa kwe-kinetics yekugcwala kwemahomoni etitjalo letibangelwa tilwane letidla titjalo. Le node imelela ihormone yesitjalo sinye, bese bukhulu be node bumele inkomba ye Si lecondzile ku hormone yesitjalo emkhatsini wekwelashwa. (B) Kugcwala kwe JA, JA-Ile kanye ne ET emacembeni lokubangelwa kwelashwa lokuhlukene lokukhonjiswe ngemibala lehlukene: i-apricot, W + OSM; luhlata sasibhakabhaka, W + OSSl; lokumnyama, W + W; mpunga, C (kulawula) . Tinkhanyeti tikhombisa umehluko lomkhulu emkhatsini wekwelashwa nekulawula (i-ANOVA yetindlela letimbili lelandzelwa kucatsanisa lokunyenti kwe-Tukey HSD post hoc, *** P <0.001). Kuhlatiya kwemcondvo welwatiso lwe (C)697 MS/MS (lifayela lemininingwane S1) ku-JA biosynthesis kanye ne-spectrum yekucabanga lekhubatekile (irAOC kanye ne-irCOI1) kanye (D)585 MS/MS (ifayela lemininingwane S1) ku-ETR1 ngekukhubateka kwesiginali ye-ET (Luhlelo lwekulawula tilwane letidla tilwane letidla tilwane letidla titjalo letimbili letilingisiwe) lwavusa. Tinkhanyeti tikhombisa umehluko lomkhulu emkhatsini wekwelashwa kwe-W + OS kanye nekulawula lokungalimali (i-ANOVA yetindlela letimbili lelandzelwa kucatsanisa lokunyenti kwe-Tukey HSD post hoc, * P <0.05, ** P <0.01 kanye *** P <0.001). (E) Emagrafu lasakatekile ekuphikiswa lokusakatekile kwekukhetsekile. Lemibala imelela tinhlobo letehlukene letiguculwe ngekwelifa; letimphawu timelela tindlela letehlukene tekwelapha: unxantathu, W + OSS1; unxande, W + OSM; umbuthano C
Ngemuva kwaloko, sisebentisa luhlobo loluguculwe ngekwelifa lwe Nepenthes lebutsakatsaka (irCOI1 na sETR1) etinyatselweni letibalulekile te JA na ET biosynthesis (irAOC na irACO) kanye nekucabanga (irCOI1 na sETR1) kuhlatiya kusebenta kwalamahomoni lamabili etitjalo kumnikelo wetilwane letidla titjalo Lesihlobo. Kuhambisana nekuhlolwa kwaphambilini, sacinisekisa kufakwa kwe herbivore-OS etitjalweni letite lutfo (EV) (Umfanekiso 3, C kuya ku D) kanye nekwehla jikelele kwenkomba ye Hj lebangelwa yi OSM, kantsi inkomba ye δj yakhula. Imphendvulo ibonakala kakhulu kunemphendvulo levuswa yi-OSS1. Igrafu lenemigca lemibili lesebentisa iHj ne δj njengetikhonjiso ikhombisa kukhishwa kwekulawula lokutsite (Umfanekiso 3E). Lokuhamba embili lokusobala kakhulu kutsi etinhlobeni letite luphawu lwe JA, kwehlukahlukana kwe metabolome kanye netingucuko tekukhetseka letibangwa tilwane letidla titjalo ticishe ticedze ngalokuphelele (Umfanekiso 3C). Ngalokwehlukile, umbono we-ET lothulile etitjalweni te-sETR1, noma umphumela jikelele etingucukweni tekugaya tilwane letidla titjalo uphansi kakhulu kunalowo we-JA signaling, unciphisa umehluko ku-Hj kanye ne-δj indices emkhatsini we-OSM kanye ne-OSS1 excitations (Umfanekiso 3D kanye neMfanekiso S5). . Loku kukhombisa kutsi ngetulu kwemsebenti lomcoka wekuguculwa kwesiginali ye JA, kuguculwa kwesiginali ye ET kuphindze kusebente njengekulungisa kahle kwemphendvulo lekhetsekile yetinhlobo tetilwane letidla titjalo. Kuhambisana nalomsebenti wekulungisa kahle, bekute lushintjo ekungenisweni kwe metabolome jikelele etitjalweni te sETR1. Ngakulolunye luhlangotsi, uma kucatsaniswa netitjalo te sETR1, titjalo te irACO tikhutsate ema amplitudes lafanako jikelele etingucuko tekugaya letibangelwa tilwane letidla titjalo, kepha takhombisa emamaki lehluke kakhulu eHj kanye ne δj emkhatsini wetinsayeya te OSM kanye ne OSS1 (Umfanekiso S5).
Kute sibone ema metabolites lakhetsekile laneminikelo lebalulekile ekuphendvuleni kwetinhlobo letikhetsekile tetilwane letidla titjalo kanye nekucolisa umkhicito wato ngetimphawu te ET, sasebentisa indlela yesakhiwo se MS/MS leyakhiwe ngaphambilini. Lendlela incike endleleni ye-bi-clustering kuphindze iphetse umndeni we-metabolic kusuka etincenyeni te-MS / MS [umkhicito wemachashazi lojwayelekile (NDP)] kanye nesikolo sekufana lokusekelwe ekulahlekelweni lokungatsatsi luhlangotsi (NL). Isethi yedatha ye-MS / MS leyakhiwe ngekuhlatiya imigca ye-ET transgenic yakhicita 585 MS / MS (ifayela ledatha S1), leyalungiswa ngekutsi ihlanganiswe ibe ngemamojula lasikhombisa lamakhulu e-MS / MS (M) (Umfanekiso 4A) . Lamanye alamamojula agcwele kakhulu ngetintfo letikhetsekile letibonakalako ngaphambilini: sibonelo, iM1, M2, M3, M4 kanye ne M7 inotsile ngetintfo letehlukene letisuselwa ku phenol (M1), ema flavonoid glycosides (M2), shukela we acyl (M3 Na M4), kanye ne 17-HGL-DTG (M7). Ngetulu kwaloko, lwati lolutsite lwe-metabolic (Si index) lwe-metabolite yinye kumojuli ngamunye luyabalwa, futsi kusabalalisa kwayo kwe-Si kungabonakala ngendlela lebonakalako. Ngamafuphi, iMS/MS spectra lekhombisa kudla kwetitjalo lokusetulu kanye ne genotype lekhetsekile ibonakala ngemanani lasetulu eSi, kanye netibalo te kurtosis tikhombisa kusabalalisa boya ekhoneni lemsila lesekudla. Lokunye lokunjalo kwekusakateka kwe colloid lean kwatfolakala ku M1, lapho khona iphenol amide yakhombisa ingcenye lephakeme ye Si (Umfanekiso 4B). Le herbivorous inducible 17-HGL-DTG leshiwo ngaphambilini ku M7 yakhombisa emaphuzu eSi lalinganiselwe, lakhombisa lizinga lelilinganiselwe lekulawula kwemehluko emkhatsini waletinhlobo letimbili te OS. Ngalokwehlukile, ema metabolites lakhetsekile lakhicitwa ngendlela lesungulako, njenge rutin, CGA, kanye ne acyl sugars, asemkhatsini wemaphuzu laphansi e Si. Kute kuhlolelwe kancono kuyinkimbinkimbi kwesakhiwo kanye nekusakateka kwe Si emkhatsini wema metabolites lakhetsekile, inethiwekhi ye molecule yakhiwa ku module ngayinye (Umfanekiso 4B). Kubiketela lokubalulekile kwe OD theory (lokufingqiwe ku Figure 1B) kutsi kuhlelwa kabusha kwema metabolites lakhetsekile ngemuva kwe herbivory kufanele kuholele ekugucukeni kwendlela yinye kuma metabolites lanelinani leliphakeme lekuvikela, ikakhulukati ngekukhulisa kucaciswa kwawo (njengobe kucatsaniswa nekusakateka lokungahleliwe) Indlela) I metabolite yekuvikela lebiketelwe yi MT theory. Liningi letintfo letisuselwa ku phenol letibutfwe ku M1 tihlobene ngekusebenta nekwehla kwekusebenta kwetinambuzane (32). Uma kucatsaniswa emanani eSi kuma metabolites eM1 emkhatsini wemacembe lakhutsatwako kanye nemacembe lakhiwe etitjalweni tekulawula te EV emahoreni langemashumi lamabili nakune, sabona kutsi kukhetseka kwe metabolites kwema metabolites lamaningi ngemuva kwetinambuzane letidla titjalo kunendlela lebalulekile yekukhula (Umfanekiso 4C). Lokukhula lokutsite kwelinani le Si kwatfolakala kuphela kuma phenolamides lavikelako, kepha kute kukhula kwelinani le Si kwatfolakala kulamanye ema phenols kanye nema metabolites langatiwa lahlala ndzawonye kule module. Lena yimodeli lekhetsekile, lehlobene ne-OD theory. Tibiketelo letiyinhloko tetingucuko tekugaya kudla letibangelwa tilwane letidla titjalo tiyahambisana. Kute sivivinye kutsi ngabe loku lokukhetsekile kwe-phenolamide spectrum kwabangelwa yi-OS-ET lekhetsekile, sakhetsa inkomba ye-metabolite Si futsi sabangela linani lekuveta umehluko emkhatsini we-OSM ne-OSS1 ku-EV ne-sETR1 genotypes (Umfanekiso 4D ). Ku-sETR1, umehluko lobangelwa yi-phenamide emkhatsini we-OSM ne-OSS1 wancishiswa kakhulu. Indlela ye-bi-clustering yaphindze yasetjentiswa kudatha ye-MS / MS lebutfwe etinhlobeni letine-JA lengakeneli kute kuphetfwe ngemamojula layinhloko e-MS / MS lahlobene nekukhetseka kwe-metabolic lokulawulwa yi-JA (Umfanekiso S6).
(A) Imiphumela yekuhlanganisa ye-585 MS / MS lesekelwe esigabeni lesabelwana ngaso (kufana kwe-NDP) kanye nekulahlekelwa lokungatsatsi luhlangotsi lokwabelwana ngako (kufana kwe-NL) kuphumela ekutsi imojuli (M) ihambisane nemndeni we-compound lowatiwako, noma ngekwakheka kwe-Metabolite lokungatiwa noma lokungagucuki kahle. Eceleni kwemojuli ngayinye, kukhonjiswa kusabalalisa kwe-metabolite (MS / MS) lokutsite (Si). (B) Inethiwekhi yemamolekhyuli e-modular: Ema-node amelela i-MS / MS kanye nemiphetho, i-NDP (lebovu) kanye ne-NL (leluhlata sasibhakabhaka) emaphuzu e-MS / MS (kusika,> 0.6). Inkomba yekucaciswa kwe metabolite lelinganisiwe (Si) lefakwe imibala ngekuya ngemojuli (ngasencele) futsi ifakwe kumephu kunethiwekhi yemamolekhyuli (ngasekudla). (C) Mojuli M1 wesitjalo se-EV esimeni sekusungulwa (kulawula) kanye nesimo lesikhutsatwako (kulingisa herbivore) emahoreni langu-24: umdvwebo wenethiwekhi yemamolekhyuli (Si linani lisayizi le-node, i-phenolamide levikelako igcizelelwa ngeluhlata sasibhakabhaka). (D) Umdvwebo we-M1 molecule inethiwekhi ye-spectrum line sETR1 ngekukhubateka kwe-EV ne-ET umbono: le-phenolic compound lemelelwa yi-node yembutsano loluhlata, kanye nemehluko lomkhulu (linani le-P) emkhatsini we-W + OSM ne-W + OSS1 yekwelashwa njengesayizi ye-Node. CP, i-N-ikhefiyoyili; CS, i-N-ikhefiyoyili-i-sipirimidini; FP, i-N-esidi ye-ester-e-uric esidi; FS, i-N-i-feruli-i-spermidine; I-CoP, N', N I-CFS, i-N', i-N”-ikhafeyoyli, i-feruloyl-spermidine;
Siphindze sandzisa kuhlatiya kusuka ku-genotype yinye ye-Nepenthes lebutsakatsaka kuya kubantfu bemvelo, lapho khona tingucuko leticinile te-intraspecific emazingeni e-JA ladla titjalo kanye nemazinga lakhetsekile e-metabolite achazwe ngaphambilini kubantfu bemvelo (26). Sebentisa lesethi yedatha kuhlanganisa emagciwane langu-43. Lama germplasms afaka tinhlobo letingu 123 tetitjalo letivela ku N. pallens. Letitjalo tatsatfwa etimbewu letibutfwe etindzaweni letehlukene temdzabu eUtah, Nevada, Arizona, nase California (Umfanekiso S7), sabala kwehlukahlukana kwe metabolome (lapha kubitwa ngekutsi lizinga lebantfu) β kwehlukahlukana) kanye nekukhetseka lokubangelwa yi OSM. Kuhambisana netifundvo letindlulile, sabona tingucuko letiningi tekugaya eceleni kwema-axes e-Hj kanye ne-δj, lokukhombisa kutsi ema-germplasms anemehluko lomkhulu epulasitikini yetimphendvulo tabo tekugaya ku-herbivores (Umfanekiso S7). Lenhlangano ikhumbuta lokubonwe ngaphambilini mayelana neluhlu lolushintjashintjako lwetingucuko tema JAs letibangelwa tilwane letidla titjalo, futsi igcine linani leliphakeme kakhulu kubantfu lababodwa (26, 36). Ngekusebentisa i-JA ne-JA-Ile kuhlola kuhambisana kwelizinga jikelele emkhatsini we-Hj ne-δj, satfola kutsi kunekuhambisana lokuhle lokubalulekile emkhatsini we-JA kanye ne-metabolome β kwehlukahlukana kanye nenkomba yekukhetsekile (Umfanekiso S7). Loku kukhombisa kutsi le heterogeneity lebangwa tilwane letidla titjalo ku JAs induction letfolakala esigabeni sebantfu kungenteka kutsi kungenca yema polymorphisms labalulekile e metabolic labangelwa kukhetsa kusuka etilwaneni letidla titjalo letinetinambuzane.
Tifundvo letindlulileko tikhombise kutsi tinhlobo teligwayi tehluke kakhulu ngeluhlobo kanye nekuncika lokuhlobene ekuvikeleni kwemetabolic lokukhutsatwako nalokusungulako. Kukholelwa kutsi letingucuko te anti-herbivore signal transduction kanye nemandla ekuvikela kulawulwa ngumfutfo webantfu betinambuzane, umjikeleto wemphilo yetitjalo, kanye netindleko tekukhicita tekuvikela kule niche lapho khona luhlobo loluniketiwe lukhula khona. Sifundze ngekuhambisana kwekuvuselelwa kwemacembe e-metabolome lokubangelwa tilwane letidla titjalo tetinhlobo letisitfupha te-Nicotiana letivela eNorth America naseNingizimu Melika. Leti tinhlobo tihlobene kakhulu ne Nepenthes yase North America, lokungu Nicolas Bociflo. La, N. ema-nikotini, ema-nikoti n. tjani lobubutsakatsaka, iNicotiana tabacum, ligwayi lelisemgceni, ligwayi (iNicotiana spegazzinii) kanye neligwayi lemacembe egwayi (iNicotiana obtusifolia) (Umfanekiso 5A) (37). Tinhlobo letisitfupha taletinhlobo, kufaka ekhatsi tinhlobo letibonakala kahle N. sicela, titjalo temnyaka te clade ye petunia, kantsi ne obtusifolia N. titjalo letihlala njalo te clade yadzadzewabo iTrigonophyllae (38). Ngemuva kwaloko, W + W, W + OSM kanye ne W + OSS1 induction kwentiwa kuletinhlobo letisikhombisa kwentela kufundza kuhleleka kabusha kwe metabolic yelizinga letinhlobo kwekudla kwetinambuzane.
(A) Sihlahla se-phylogenetic se-bootstrap lesisekelwe ematfuba lamakhulu [ekuhlanganiswa kwe-glutamine yenuzi (38)] kanye nekusakateka kwemhlaba kwetinhlobo letisikhombisa te-Nicotiana letihlobene kakhulu (imibala leyehlukene) (37). (B) Indzawo yekusakateka yekuhlukahluka lokukhetsekile kwemaphrofayli ekugaya etinhlobo letisikhombisa teNicotiana (939 MS / MS; ifayela lemininingwane S1). Esigabeni setinhlobo, kwehlukahlukana kwe metabolome kuhambisana kabi nelizinga lekukhetsekile. Kuhlatiya kwekuhambisana kwetinhlobo-telizinga emkhatsini wekuhlukahlukana kwemetabolic kanye nekukhetseka kanye nekugcwala kwe JA kukhonjisiwe ku Figure 2. S9. Umbala, tinhlobo letehlukene; unxantathu, W + OSS1; unxande, W + OSM; (C) Nicotiana JA kanye ne-JA-Ile Dynamics ibekwe ngekuya kwe-OS kujabula kwemandla (i-ANOVA yetindlela letimbili kanye ne-Tukey HSD ngemuva kwekucatsanisa lokunyenti, * P <0.05, ** P <0.01 kanye * ** Kucatsanisa W + OS kanye ne-W + W, P <0.001). Libhokisi lendzawo ye (D) kwehlukahlukana kanye (E) nekukhetseka kweluhlobo ngalunye ngemuva kwekulingisa tilwane letidla titjalo kanye ne methyl JA (MeJA). Lenkhanyeti ikhombisa umehluko lomkhulu emkhatsini we W + OS ne W + W noma i-lanolin kanye ne W (Lan + W) noma iLan kanye ne MeJA (Lan + MeJa) kanye nekulawula kwe Lan (kuhlatiya kwetindlela letimbili tekuhluka, kulandzelwe kucatsanisa lokunyenti kwe-HSD yaTukey ngemuva kwe-hoc , * P <0.05, ** P <0.01 kanye).
Kusebentisa indlela ye-cluster lembili, sakhomba emamojula langu-9 e-939 MS / MS (ifayela lemininingwane S1). Kubunjwa kwe MS/MS lelungiswe kabusha ngetindlela tekwelapha letehlukene kuyahluka kakhulu emkhatsini wemamojula lehlukene emkhatsini wetinhlobo (Umfanekiso S8). Kubona ngemehlo engcondvo iHj (lebitwa lapha ngekutsi yi-species-level γ-diversity) kanye ne δj kuveta kutsi tinhlobo letehlukene tihlangana tibe ngemacembu lehluke kakhulu esikhaleni sekugaya, lapho khona kwehlukana kwetinhlobo-level kuvame kuvela kakhulu kunekujabula. Ngaphandle kwe N. linear kanye ne N. obliquus, tikhombisa tinhlobo letiningi letishintjashintjako temiphumela yekungeniswa (Umfanekiso 5B). Ngalokwehlukile, tinhlobo letifana ne N. purpurea kanye ne N. obtusifolia tinekuphendvula lokuncane kwekugaya kudla ekwelashweni, kepha imetabolome ihlukahlukene kakhulu. Kusabalaliswa kwetinhlobo letikhetsekile tekuphendvula kwekugaya lokubangelwako kwaholela ekuhambisaneni lokubi lokubalulekile emkhatsini wekukhetseka kanye nekuhlukahluka kwe gamma (PCC = -0.46, P = 4.9 × 10-8). Tingucuko letibangwa yi-OS emazingeni e-JAs tihambisana kahle nekukhetseka kwe-metabolome, futsi tihambisana kabi nekuhlukahluka kwe-metabolic gamma lokukhonjiswa luhlobo ngalunye (Umfanekiso 5B kanye neMfanekiso S9). Kufanele kutsi kucashelwe kutsi letinhlobo letibitwa ngesilulumagama ngekutsi “tinhlobo tekuphendvula kwesiginali” kuMfanekiso 5C, njenge Nepenthes nematodes, Nepenthes Nepenthes, Nepenthes acute, kanye ne Nepenthes lebutsakatsaka, kwabangela timphawu letibalulekile ngemizuzu lengemashumi lamatsatfu. Lokubhubha kwe JA kanye ne JA-Ile lekhetsekile ye OS kwamuva, kantsi lamanye emagciwane labitwa ngekutsi “asibonakaliso lesingaphendvuli”, njengetigayini te Nepenthes, iNepenthes lempuphu kanye ne N. Esigabeni se-metabolic, njengoba kushiwo ngenhla, ku-Nepenthes lebutsakatsaka, tintfo letiphendvula luphawu takhombisa kucaciswa kwe-OS futsi takhulisa kakhulu i-δj, ngesikhatsi tinciphisa i-Hj. Lomphumela wekucala we-OS-lokhetsekile awuzange utfolakale etilwaneni letihlukaniswe njengetinhlobo letingaphendvuli tesiginali (Umfanekiso 5, D na E). Ema-metabolites lakhetsekile e-OS abelana kakhulu emkhatsini wetinhlobo letiphendvula luphawu, futsi lamacembu eluphawu ahlangana netinhlobo letinetimphendvulo letibutsakatsaka teluphawu, kantsi tinhlobo letinetimphendvulo letibutsakatsaka teluphawu tikhombisa kuncika lokuncane (Umfanekiso S8 ). Lomphumela ukhombisa kutsi kufaka i-OS-lekhetsekile yema JAs kanye nekuhleleka kabusha kwe OS-lekhetsekile ye metabolome lesezansi kuhlanganiswa ezingeni letinhlobo.
Ngemuva kwaloko, sasebentisa i-lanolin paste lecuketse i-methyl JA (MeJA) kwelapha titjalo kuphenya kutsi ngabe letindlela tekuhlanganisa tivinjelwe yini ngekutfolakala kwe-JA lesetjentiswa yi-exogenous JA, letawuba ku-cytoplasm yetitjalo. Kukhishwa kwe-ester ngekushesha yi-JA. Sitfole umkhuba lofanako wekushintja kancane kancane kusuka etilwaneni letiphendvula luphawu kuya etilwaneni letingaphendvuli ngeluphawu lokubangelwa kuniketwa lokuchubekako kwe JA (Umfanekiso 5, D na E). Ngamafuphi, kwelashwa kwe MeJA kwahlela kabusha ngamandla ema metabolomes ema nematodes lasemgceni, i N. obliquus, i N. aquaticus, i N. N. purpurea wakhombisa kuphela kukhula kwe δj, kepha hhayi Hj. iN.obtusifolia, leseyikhonjiswe ngaphambilini kutsi ibutsela ndzawonye emazinga laphansi kakhulu ema JAs, nayo ayiphendvuli kahle ekwelashweni kwe MeJA ngekuya kwekucubungula kabusha kwe metabolome. Lemiphumela ikhombisa kutsi kukhicita kwe JA noma kuguculwa kwesiginali kuvinjelwe ngekwemtimba etilwaneni letingaphendvuli kwesiginali. Kuvivinya lomcabango, safundza tinhlobo letine (N. pallens, N. tigayo, N. pink kanye ne N. microphylla) letikhutsatwa yi W + W, W + OSMs kanye ne W + OSS1 Transcriptome (39). Kuhambisana nephethini yekulungisa kabusha kwe metabolome, letinhlobo tehlukaniswe kahle esikhaleni se transcriptome, emkhatsini lapho khona iN. attenuated yakhombisa iRDPI lesetulu lebangelwa yi OS, kantsi iN. Nomakunjalo, kwatfolakala kutsi kwehlukahlukana kwe transcriptome lokubangwa yi N. oblonga bekuphansi kakhulu kuletinhlobo letine, ngekuphikisana nekuhlukahluka lokusetulu kwe metabonomic ye N. oblonga lebeyikhonjiswe ngaphambilini etilwaneni letisikhombisa. Tifundvo letindlulileko tikhombise kutsi isethi yemajini lahlobene netimphawu tekuvikela tekucala, kufaka ekhatsi timphawu te JA, ichaza kucaciswa kwetimphendvulo tekuvikela tekucala letibangwa ngema elicitors lahlobene ne herbivore etilwaneni te Nicotiana (39). Kucatsanisa tindlela tekukhombisa te JA emkhatsini waletinhlobo letine kwaveta iphethini lejabulisako (Umfanekiso 6B). Emajini lamaningi kulendlela, njenge AOC, OPR3, ACX kanye ne COI1, akhombisa emazinga laphakeme ekufakwa kuletinhlobo letine. Nomakunjalo, ligciwane lelibalulekile, iJAR4, igucula iJA ibe yindlela yayo lesebentako yetilwane ye JA-Ile lebhalwe phansi lebutfwe, futsi lizinga layo lekubhala liphansi kakhulu, ikakhulu ku N. mills, Nepenthes pieris kanye ne N. microphylla. Ngetulu kwaloko, kuphela lokubhalwe phansi kwalelinye lifutsi AOS akuzange kutfolakale ku N. bifidum. Letingucuko ekuboniseni emajini tingase tibe nesibopho se phenotypes lembi kakhulu lebangelwa kukhicita lokuphansi kwe JA etilwaneni te signal anergic kanye nekufakwa kwe N. gracilis.
(A) Kuhlatiya kwemcondvo welwati lwekuhlela kabusha kwetimphendvulo tekubhala tekucala tetinhlobo letine teligwayi letihlobene kakhulu letisampuliwe ngemuva kwemizuzu lengu-30 ngemuva kwekungeniswa kwe-herbivory. iRDPI ibalwa ngekucatsanisa emacembe labangelwa yi herbivore OS nekulawula silonda. Imibala ikhombisa tinhlobo letehlukene, bese timphawu tikhombisa tindlela letehlukene tekwelapha. (B) Kuhlatiya kuveta ufuto etindleleni tekukhombisa te-JA emkhatsini wetinhlobo letine. Indlela ye-JA leyentiwe lula ikhonjiswa eceleni kwesakhiwo sebhokisi. Imibala leyehlukene ikhombisa tindlela letehlukene tekucubungula. Inkhanyeti ikhombisa kutsi kunemehluko lomkhulu emkhatsini wekwelashwa kwe-W + OS kanye nekulawula kwe-W + W (kwe-t-test yeMfundzi yemehluko wekubhaca, * P <0.05, ** P <0.01 na *** P <0.001). OPDA, i-esidi le-12-ye-oxophytodienoic; I-OPC-8: 0,3-oxo-2 (2'(Z)-iphentinayili)-i-cyclopentane-1-esidi ye-oktanoyiki.
Esigabeni lekugcina, safundza kutsi kuvuselelwa kabusha kwetinhlobo tetinambuzane-letitsite kwe metabolome yetinhlobo letehlukene tetitjalo kungamelana njani netilwane letidla titjalo. Lucwaningo lwangaphambilini lwagcizelela luhlobo lwe Nicotiana. Kumelana kwawo ne Ms kanye netibungu kwehluka kakhulu (40). Lapha, safundza kuchumana emkhatsini wale modeli kanye plasticity yabo metabolic. Kusebentisa letinhlobo letine tegwayi letingenhla, kanye nekuhlola kuhambisana emkhatsini wekuhlukahluka kanye nekukhetseka kwe metabolome lokubangelwa tilwane letidla titjalo kanye nekumelana kwetitjalo ku Ms na Sl, satfola kumelana, kwehlukahlukana kanye nekukhetseka ku generalist Sl Konkhe kuhambisana kahle, kantsi kuhambisana emkhatsini wekuhlukahlukana lokukhetsekile kwe ladies hhayi nge Fig S10). Mayelana nekumelana ne S1, kokubili le N. chinensis lebutsakatsaka kanye ne N. gracilis, lebeyikhonjiswe ngaphambilini kutsi ikhombisa kokubili emazinga ekugucula luphawu lwe JA kanye ne metabolome plasticity, beyinetimphendvulo letehluke kakhulu ekungenisweni kwetilwane letidla titjalo, futsi nato takhombisa kumela lokusetulu lokufanako. Kuya ecasini.
Eminyakeni lengemashumi lasitfupha leyengcile, indlela yekuvikela titjalo inikete luhlaka lwetifundvo, lolusekelwe lapho khona bacwaningi babiketele linani lelikhulu lekutigucukela kwemvelo kanye nemisebenti yetitjalo letikhetsekile te metabolites. Liningi laletifundvo alilandzeli inchubo levamile yekuphetsa lokucinile (41). Baphakamisa kucagela lokubalulekile (3) ezingeni lelifanako lekuhlatiya. Lapho kuhlolwa kwekubiketela lokubalulekile kuvumela kutsi kuhlaziywe imibono letsite, loku kutawenta kutsi Insimu iyachubeka. Usekelwe, kepha ulahle labanye (42). Esikhundleni saloko, le theory lensha yenta kucagela emazingeni lehlukene ekuhlatiya futsi yengeta lucwencwe lolusha lwekucabanga lokuchazako (42). Kepha, letifundvo letimbili letiphakanyisiwe ezingeni lekusebenta, i-MT ne-OD, ingachazwa kalula njengekubiketela lokubalulekile kwetingucuko letikhetsekile tekugaya kudla letibangelwa tilwane letidla titjalo: i-OD theory ikholelwa kutsi tingucuko letikhetsekile tekugaya kudla “esikhaleni” ticondziswe kakhulu . Le MT theory ikholelwa kutsi letingucuko titawube tingacondzi futsi tibe khona ngekungahleleki esikhaleni sekugaya, futsi tivame kuba nema metabolites lasetulu ekuvikela. Kuhlolwa kwangaphambilini kwekubiketela kwe-OD kanye ne-MT kuhlolwe kusetjentiswa isethi lencane yetinhlanganisela te-priori “tekuvikela”. Letivivinyo letisemkhatsini we metabolite tivimbela likhono lekuhlatiya lizinga kanye nemzila wekuhlelwa kabusha kwe metabolome ngesikhatsi se herbivory, futsi ativumeli kuhlolwa ngaphakatsi kweluhlaka lwetibalo loluhambisanako kutsi kudzingeke kucagela lokubalulekile lokungatsatfwa njengekuphelele Kulinganisa tingucuko ku metabolome yesitjalo. Lapha, sasebentisa tebuchwepheshe lobusha ku-metabolomics lesekelwe ku-MS yekubala futsi senta kuhlatiya kwe-deconvolution MS emalini jikelele yetinchazelo tetifundvo telwati kute sivivinye umehluko emkhatsini walaba lababili labaphakanyisiwe ezingeni le-metabolomics lemhlaba wonkhe. Kubiketela lokubalulekile kwale theory. Ithiyori yelwati isetjentiswe etindzaweni letiningi, ikakhulukati esimeni setinhlobonhlobo tetilwane kanye nelucwaningo lwekugeleta kwemanutrients (43). Nomakunjalo, ngekwati kwetfu, lesi sicelo sekucala lesisetjentiswa kuchaza sikhala selwati lwe metabolic yetitjalo kanye nekusombulula tinkinga temvelo letihlobene netingucuko tesikhashana te metabolic ngekuphendvula timphawu temvelo. Ikakhulu, likhono lalendlela lilele emandleni ayo ekucatsanisa emaphethini ngekhatsi kanye nasemkhatsini wetinhlobo tetitjalo kuhlola kutsi tilwane letidla titjalo tigucuke njani kusuka etilwaneni letehlukene kuya kumaphethini e-inter-species macroevolutionary emazingeni lehlukene ekugucuka. Kugaya kudla.
Kuhlatiya tincenye letiyinhloko (PCA) kugucula isethi yedatha leyehlukahlukene ibe sikhala sekunciphisa bukhulu kute kutsi umkhuba lomkhulu wedatha ungachazwa, ngako-ke uvame kusetjentiswa njengendlela yekuhlola yekuhlatiya isethi yedatha, njenge-deconvolution metabolome. Nomakunjalo, kuncishiswa kwebukhulu kutawulahlekelwa yincenye yelwati lolucuketfwe kusethi yedatha, futsi iPCA ayikhoni kuniketa lwati lwesilinganiso mayelana netimphawu letihambisana kakhulu ne theory yemvelo, njenge: kutsi tilwane letidla titjalo tilungisa njani kwehlukahlukana emasimini lakhetsekile (sibonelo, kuceba, kusabalalisa) Kanye nebuningi) ema metabolites? Ngutiphi ti-metabolites letibiketela simo lesikhutsatwako se-herbivore leniketiwe? Kusuka kumbono wekukhetsa, kwehlukahlukana kanye nekungatsatfwa, lokucuketfwe kwelwati lwephrofayli ye metabolite lekhetsekile yemacembe kuyabola, futsi kutfolakala kutsi kudla kwetilwane letidla titjalo kungenta kutsi kusebente indlela lekhetsekile yekugaya kudla. Ngekungalindzeli, sabona kutsi, njengoba kuchaziwe etikhombisweni tetifundvo telwatiso letiphunyelelisiwe, simo sekugaya lokuphumelako sinekuhlangana lokukhulu ngemuva kwekuhlaselwa kwetilwane letimbili letidla titjalo (i-generalist ledliwa ebusuku Sl) kanye nengcweti yeSolanaceae Ms. Noma nje kutiphatsa kwabo kwekudla kanye nekugcila kwehluke kakhulu. Umsunguli we-esidi yemafutsa-esidi ye-amino (FAC) ku-OS (31). Ngekusebentisa i-OS yekudla titjalo kwelapha tilondza tekubhoboka letijwayelekile, kwelashwa kwekudla kwetitjalo lokulingisiwe nako kwakhombisa umkhuba lofanako. Lenchubo lesemgangatfweni yekulingisa kuphendvula kwetitjalo ekuhlaselweni kwetilwane letidla titjalo isusa tintfo letididako letibangelwa tingucuko ekutiphatseni kwekudla kwetilwane letidla titjalo, lokuholela ekulimaleni lokuhlukahlukene ngetikhatsi letehlukene (34). I-FAC, leyatiwa kutsi yimbangela lenkhulu ye-OSM, inciphisa i-JAS kanye naletinye timphendvulo temahomoni esitjalo ku-OSS1, kantsi i-OSS1 inciphisa emakhulu etikhatsi (31). Kepha, i-OSS1 yabangela emazinga lafanako ekugcwala kwe-JA uma kucatsaniswa ne-OSM. Kukhonjisiwe ngaphambilini kutsi imphendvulo ye JA ku Nepenthes lencishisiwe iyabucayi kakhulu ku OSM, lapho khona iFAC ingagcina umsebenti wayo noma ngabe ihlanjululwe 1:1000 ngemanti (44). Ngako-ke, uma kucatsaniswa ne-OSM, noma i-FAC ku-OSS1 iphansi kakhulu, yanele kutsi ikhutsate kubhubha kwe-JA lokwenele. Tifundvo letindlulileko tikhombise kutsi emaprotein lafana ne porin (45) kanye ne oligosaccharides (46) angasetjentiswa njengetinkhomba tema molecule kwentela kuvusa timphendvulo tekuvikela titjalo ku OSS1. Kepha, solo akucaci kutsi lama-elicitors ku-OSS1 anesibopho sekugcwala kwe-JA lebonwe kulolucwaningo lwanyalo.
Noma kunetifundvo letimbalwa letichaza umehluko weminwe yekugaya kudla lokubangelwa kusetjentiswa kwetilwane letidla titjalo letehlukene noma i-exogenous JA noma i-SA (salicylic acid) (47), kute lophazamise kuphazamiseka lokukhetsekile kwetinhlobo tetilwane letidla titjalo kunethiwekhi yetjani besitjalo kanye nemiphumela yayo ku-metabolism lekhetsekile Le study jikelele. lwati loluphatselene nawe. Lokuhlatiya kwaphindze kwacinisekisa kutsi kuchumana kwenethiwekhi yemahomoni langekhatsi nalamanye emahomoni etitjalo ngaphandle kwema JAs kubumba kukhetseka kwekuhlelwa kabusha kwe metabolic lokubangelwa tilwane letidla titjalo. Ikakhulukati, satfola kutsi i-ET lebangelwa yi-OSM beyinkulu kakhulu kunaleyo lebangelwa yi-OSS1. Lendlela ihambisana nekucuketfwe kwe-FAC lokunyenti ku-OSM, lokusimo lesidzingekako nalesenele sekuvusa kuqhuma kwe-ET (48). Ngesimo sekusebentisana emkhatsini wetitjalo kanye netilwane letidla titjalo, umsebenti wekukhombisa we ET ku metabolite dynamics lekhetsekile yetitjalo solo uyahamba ngetikhatsi letitsite futsi uhlose licembu linye lelihlangene kuphela. Ngekwengeta, tifundvo letiningi tisebentise kusetjentiswa kwe exogenous kwe ET noma tintfo letihamba embili noma tivimbeli letehlukene kufundza kulawulwa kwe ET, emkhatsini wato leticelo temakhemikhali exogenous titawukhicita imiphumela leminyenti lengakakhetsi. Ngekwati kwetfu, lolucwaningo lumelele kuhlolwa kwekucala lokukhulu lokuhlelekile kwendzima ye-ET ekusetjentisweni kwe-ET kukhicita kanye nekubona titjalo letikhubatekile te-transgenic kuhlanganisa i-metabolome yetitjalo. Kufakwa kwe ET lekhetsekile ye Herbivore kungagcina kugucula imphendvulo ye metabolome. Lokubaluleke kakhulu kuphatfwa kabi kwe ET biosynthesis (ACO) kanye nekucabanga (ETR1) kwemajini laveta kuhlanganiswa kwe de novo lekhetsekile yetilwane letidla titjalo te phenolamides. Kuye kwakhonjiswa ngaphambilini kutsi i-ET ingakhona kulungisa kahle kuhlanganiswa kwe-nicotine lebangelwa yi-JA ngekulawula i-putrescine N-methyltransferase (49). Kepha, kusuka ekubukeni kwemishini, akucaci kahle kutsi i-ET ilungisa njani kungeniswa kwe-phenamide. Ngekwengeta emsebentini wekugucula luphawu lwe-ET, umfutfo wekugaya ungaphindze ushintshelwe ku-S-adenosyl-1-methionine kulawula lutjalomali kuma-amides e-polyaminophenol. S-adenosyl-1-methionine ngu ET kanye nendlela lesemkhatsini levamile yendlela yekukhicita tintfo letiphilako. Indlela lapho ET signal ilawula khona lizinga le-phenolamide idinga kufundvwa kabanti.
Sikhatsi lesidze, ngenca yelinani lelikhulu le metabolites lekhetsekile yesakhiwo lesingatiwa, kunakwa lokukhulu kwetigaba letitsite te metabolic akuzange kukhone kuhlola ngalokucinile tingucuko tesikhatsi tekuhlukahluka kwe metabolic ngemuva kwekusebentisana kwetilwane. Kwanyalo, ngekuya ngekuhlatiya kwe theory yelwati, umphumela lomkhulu wekutfola iMS/MS spectrum lesekelwe kuma metabolites langakhetsi kutsi tilwane letidla titjalo letidla noma letilingisa tilwane letidla titjalo tiyachubeka nekunciphisa kwehlukahlukana kwe metabolic jikelele kwe metabolome yemacembe ngesikhatsi tikhulisa lizinga layo lekukhetseka. Lokukhula kwesikhashana kwekucaciswa kwe metabolome lokubangelwa tilwane letidla titjalo kuhambisana nekukhula kwekusebentisana kwekucaciswa kwe transcriptome. Sici lesifaka sandla kakhulu kulokukhetseka lokukhulu kwe metabolome (kuba nelinani leliphakeme le Si) yi metabolite lekhetsekile lenemsebenti we herbivorous lochazwe ngaphambilini. Le modeli ihambisana nekubiketela kwe-OD theory, kepha kubiketela kwe-MT lokuhlobene nekungahleleki kwekuhlela kabusha kwe-metabolome akuhambisani. Kepha, lemininingwane iphindze ihambisane nekubiketela kwemodeli lehlanganisiwe (i-MT lencono kakhulu; Umfanekiso 1B), ngoba lamanye ema-metabolites langakachazwa lanemisebenti yekuvikela lengatiwa angase alandzelele kusabalalisa kwe-Si lokungahleliwe.
Iphethini lephawulekako lephindze yabanjwa ngulolucwaningo kutsi kusuka esigabeni se-micro-evolution (sitjalo sinye kanye nelinani legwayi) kuya esikalini lesikhulu sekutigucukela kwemvelo (tinhlobo teligwayi letihlobene kakhulu), emazinga lehlukene enhlangano yekutigucukela kwemvelo ase “kuvikeleni lokuhle kakhulu” Kukhona umehluko lomkhulu emandleni etilwane letidla titjalo. Moore kanye nalabanye. (20) kanye na Kessler na Kalske (1) ngekutimela baphakamisa kutsi kuguculwe emazinga lamatsatfu lasebentako etinhlobonhlobo tetilwane ekucaleni lahlukaniswa ngu Whittaker (50) abe tingucuko tesikhatsi letisungulako naletikhutsatwako tekuhlukahlukana kwemakhemikhali; laba babhali abazange bafinyete Tinchubo tekugcogca idatha ye-metabolome ngesikali lesikhulu nato atichazi kutsi ungabala njani kwehlukahlukana kwe-metabolic kusuka kulemininingwane. Kulolucwaningo, kulungiswa lokuncane kwekuhlukaniswa kwemisebenti yaWhittaker kutawucabanga ngekuhlukahluka kwe-α-metabolic njengekuhlukahlukana kwe-MS/MS spectra esitjalweni lesiniketwe sona, kanye nekuhlukahlukana kwe-β-metabolic njengekuhlukahlukana kwe-intraspecific lokusisekelo kwelicembu lebantfu Sikhala, kanye nekuhlukahlukana kwe-γ-metaboly kutawuba kwandziswa kwekufana kwe-analysis.
Lesibonakaliso se JA sibalulekile etimpendvulweni letiningi tekugaya tilwane letidla titjalo. Kepha, kukhona kushoda kwekuhlolwa kwesilinganiso lokucinile kweligalelo lekulawula kwe-intraspecific kwe-JA biosynthesis ekuhlukahlukeni kwe-metabolome, nekutsi ngabe luphawu lwe-JA luyindzawo levamile yekuhlukahlukana kwe-metabolic lokubangelwa kucindzeteleka esikalini lesiphakeme se-macroevolutionary kusakhonakala. Sibonile kutsi simo sekudla titjalo se Nepenthes herbivorous sikhutsata kukhetseka kwe metabolome kanye nekuhlukahluka kwekukhetseka kwe metabolome ngaphakatsi kwebantfu betinhlobo te Nicotiana kanye nasemkhatsini wetinhlobo te Nicotiana letihlobene kakhulu kuhambisana kahle ngendlela lehlelekile nekukhombisa kwe JA. Ngekwengeta, uma luphawu lwe JA lukhubatekile, kucaciswa kwe metabolic lokubangelwa yi genotype yinye herbivore kutawukhanselwa (Umfanekiso 3, C na E). Njengoba tingucuko te metabolic spectrum yebantfu be Nepenthes lababutsakatsaka ngemvelo tiningi kakhulu, tingucuko tekuhlukahluka kwe metabolic β kanye nekucaciswa kulokuhlatiya kungabangelwa kakhulu kujabula lokucinile kwetigaba tema compound lacebile nge metabolite. Lamakilasi lahlanganisiwe abusa incenye yephrofayli ye metabolome futsi aholela ekuhambisaneni lokuhle netimphawu te JA.
Ngenca yekutsi tindlela te biochemical tetinhlobo teligwayi letihlobene kakhulu nato tehluke kakhulu, ema metabolites akhonjwa ngalokukhetsekile esicini sekulinganisa, ngako-ke kuhlatiya kakhulu. Kucubungula kwe theory yelwatiso yephrofayli yekugaya kudla lebanjwe kuveta kutsi kufaka titjalo letidla titjalo kukhulisa kuhwebelana emkhatsini wekuhlukahlukana kwemetabolic gamma kanye nekukhetseka. Siginali ye-JA idlala indzima lesemkhatsini kulokuhweba. Kukhula kwekukhetseka kwe metabolome kuhambisana nekubiketela lokuyinhloko kwe OD futsi kuhambisana kahle ne JA signal, kantsi JA signal ihambisana kabi nekuhlukahluka kwe metabolic gamma. Letimodeli tikhombisa kutsi likhono le OD letitjalo lincunywa kakhulu yi plasticity ye JA, noma ngabe ngesikali se microevolutionary noma ngesikali lesikhulu sekutigucukela kwemvelo. Kuhlolwa kwekusebentisa i-JA ye-Exogenous legwema emaphutsa e-JA biosynthesis kuphindze kuvete kutsi tinhlobo teligwayi letihlobene kakhulu tingahlukaniswa tibe tinhlobo letiphendvula ngesiginali kanye naletingaphendvuli ngesiginali, njengendlela yabo ye JA kanye ne metabolome plasticity lebangwa tilwane letidla titjalo. Tinhlobo te Signal letingaphendvuli atikhoni kuphendvula ngenca yekungakhoni kwato kukhicita iJA lengekhatsi ngako-ke tingaphansi kwemikhawulo yemtimba. Loku kungabangelwa kugucuka kwalamanye emajini labalulekile endleleni yekukhombisa iJA (AOS na JAR4 ku N. crescens) ye. Lomphumela ugcizelela kutsi lamaphethini e-interspecies macroevolutionary angahle acondziswe kakhulu tingucuko ekuboneni kwemahomoni langekhatsi kanye nekuphendvula.
Ngekwengeta ekusebentisaneni emkhatsini wetitjalo kanye netilwane letidla titjalo, kuhlolwa kwekuhlukahlukana kwe metabolic kuhlobene nayo yonkhe intfutfuko lebalulekile ye theoretical elucwaningweni lekuvumelana netilwane nemvelo kanye nekuvela kwetimphawu letiyinkimbinkimbi te phenotypic. Ngekwandza kwelinani lemininingwane letfolakala ngetintfo tekusebenta tesimanje te-MS, kuhlolwa kwe-hypothesis ngekuhlukahlukana kwe-metabolic manje kungadlula umehluko we-metabolite ngamunye/wesigaba futsi kwente kuhlatiya kwemhlaba wonkhe kute kuvetwe emaphethini langalindzelekile. Enchubeni yekuhlatiya ngesikali lesikhulu, singatfo lesibalulekile ngumcondvo wekukhulelwa emabalave lanenshokutsi langasetjentiswa kuhlola idatha. Ngakoke, umphumela lomcoka wekuhlanganiswa kwanyalo kwe MS/MS metabolomics lengakhetsi kanye ne theory yelwati kutsi iniketa imethrikhi lelula lengasetjentiswa kwakha emabalave ekuphenya kwehlukahlukana kwe metabolic etikalini letehlukene te taxonomic. Kuyinto imfuneko lesisekelo lendlela. Kufundza ngekutigucukela kwemvelo kwetintfo letincane/letikhulu kanye nemvelo yemmango.
Esigabeni se macro-evolutionary, umgogodla wetjalo-tinambuzane co-evolution theory ya Ehrlich na Raven (51) kucagela kutsi kwehlukahlukana kwekuhlukahlukana kwekugaya kwetinhlobo letihlukene kuyimbangela yekuhlukahlukana kwetinhlavu tetitjalo. Kepha, eminyakeni lengemashumi lasihlanu kusukela kwashicilelwa lomsebenti we-seminal, lomcabango awukavami kuhlolwa (52). Loku kungenca kakhulu yetimphawu te phylogenetic tetimphawu tekugaya letifananako kuyoyonkhe imigca yetitjalo letisemabanga lamadze. Le rarity ingasetjentiswa kufaka tindlela tekuhlatiya letihlosiwe. Lokusebenta kwe-MS/MS kwanyalo lokucubungulwa yi-theory yelwati kulinganisa kufana kwesakhiwo se-MS/MS se-metabolites lengatiwa (ngaphandle kwekukhetsa kwe-metabolite ngaphambilini) bese kugucula lama-MS/MS abe yisethi ye-MS/MS, ngaleyo ndlela ku-metabolism yebungcweti Letimodeli te-macro-evolutionary tiyacatsaniswa ngesikali sekuhlukanisa. Tinkhomba tetibalo letilula. Lenchubo ifana nekuhlatiya kwe phylogenetic, lokungasebentisa kulandzelana kwekulandzelana kwentela kulinganisa lizinga lekuhlukahluka noma kugucuka kwetinhlavu ngaphandle kwekubiketela ngaphambilini.
Esigabeni se biochemical, umcabango wekuhlola wa Firn na Jones (53) ukhombisa kutsi kwehlukahlukana kwe metabolic kugcinwa emazingeni lehlukene kuniketa tintfo letiluhlata kwenta imisebenti yetintfo letiphilako te metabolites lebeyingakahlobani noma lefakwe esikhundleni sayo ngaphambilini. Tindlela tetifundvo telwatiso tiniketa luhlaka lapho khona letingucuko tekutigucukela kwemvelo letikhetsekile te-metabolite letenteka ngesikhatsi sekukhetseka kwe-metabolite tingalinganiswa njengencenye yenchubo yekuhlola yekutigucukela kwemvelo lephakanyisiwe: kugucukela lokusebenta ngekwetilwane kusuka ekucaciseni lokuphansi kuya ekucaciseni lokusetulu Ema-metabolites lavinjelwe endzaweni leniketiwe.
Konkhe loku, emalangeni ekucala e molecular biology, tifundvo letibalulekile tekuvikela titjalo tatfutfukiswa, futsi tindlela leticondziswa yi deductive hypothesis titsatfwa kakhulu njengendlela kuphela yenchubekelembili yesayensi. Loku kungenca kakhulu yemikhawulo yebuchwepheshe yekukala yonkhe i-metabolome. Noma tindlela leticondziswa yi-hypothesis tilusito kakhulu ekukhetseni letinye tindlela tekubangela, likhono lato lekuchubela embili kuvisisa kwetfu ngemanethiwekhi e-biochemical lilinganiselwe kakhulu kunetindlela tekubala letikhonako nyalo kusayensi yesikhatsi lesiphila kuso lesebentisa idatha. Ngako-ke, imibono lengeke ibiketelwe ingetulu kakhulu kwebubanti bemininingwane lekhonako, ngako-ke ifomula/umjikeleto wekuhlola wekucabanga wenchubekelembili emkhakheni welucwaningo angeke ucinywe (4). Sibona kusengaphambili kutsi kusebenta kwekubala kwe-metabolomics leyetfulwe lapha kungavuselela inshisekelo etindzabeni takamuva (njani) kanye nekugcina (kungani) tekuhlukahluka kwe-metabolic, futsi kube neligalelo esikhatsini lesisha sesayensi yedatha lecondziswa ngekwetifundvo. Lesikhatsi saphindze sahlola imibono lebalulekile leyaphefumulela titukulwane letengcile.
Kudla lokucondzile kwetilwane letidla titjalo kwentiwa ngekukhulisa sibungu sesibili se instar noma sibungu se Sl ecembeni linye lesitjalo se pitcher lesinembala lophaphathekile lesitjalo sinye lesiqhakazako se rose, ngekuphindzaphindza kwesitjalo lesilishumi ngesitjalo ngasinye. Letibungu tetinambuzane betiboshwa ngema clamps, bese ticubu temacembe letisele betibutfwa ngemuva kwemahora lamashumi lamabili nakune kanye na 72 ngemuva kwekutseleleka bese tiyashesha-kufriziwe, bese ema metabolites ayakhishwa.
Lingisa kwelashwa kwetilwane letidla titjalo ngendlela levumelanisiwe kakhulu. Indlela kusebentisa emasondvo ephethini yendvwangu kubhobota imigca lemitsatfu yemanyeva ngakulolunye luhlangotsi lwe midrib yemacembe lamatsatfu lanwebeke ngalokuphelele esitjalweni ngesikhatsi sesigaba sekukhula kwe garland yendvwangu, bese ngekushesha ufaka 1:5 Diluted Ms. Noma usebentise iminwe lenemagilavu ​​kubhobota iS1 wound ku. Vuna bese ucubungula licembe njengoba kuchaziwe ngenhla. Sebentisa indlela lechazwe ngaphambilini kukhipha ema metabolites layinhloko kanye nemahomoni esitjalo (54).
Kusetjentiswa kwe JA lephuma ngaphandle, lamacembe lamatsatfu e petiole alesitjalo ngasinye setitjalo letisitfupha letiqhakazako te rose yeluhlobo ngalunye alashwa nge 20μl ye lanolin lecuketse 150μg we MeJA (Lan + MeJA), kanye na 20μl we lanolin kanye nekwelapha silonda (Lan + la2), noma kusebentisa pur μe W. Emacembe avunwa ngemuva kwemahora langu 72 ngemuva kwekwelashwa, asheshe afriziwe ku nitrogen lengemanti, bese agcinwa ku -80°C kuze kube ngulapho asetjentiswa.
Imigca lemine ye-JA kanye ne-ET leguculwe, lokuyi-irAOC (36), i-irCOI1 (55), i-irACO kanye ne-sETR1 (48), seyikhonjwe ecenjini letfu lelucwaningo. irAOC ngamandla wakhombisa kwehla emazingeni JA kanye JA-Ile, kantsi irCOI1 bekungakacabangi JAs. Nawucatsanisa ne-EV, kugcwala kwe-JA-Ile kwandza. Ngendlela lefanako, i-irACO itawunciphisa kukhicitwa kwe-ET, futsi uma kucatsaniswa ne-EV, i-sETR1, lengaphatseki nge-ET, itawukhulisa kukhicitwa kwe-ET.
I-spectrometer ye-laser ye-photoacoustic (Inzwa ye-ET yesikhatsi sangempela ye-ETD-300) isetjentiselwa kwenta kukala kwe-ET ngendlela lengahlaseli. Ngekushesha ngemuva kwekwelashwa, hhafu wemacembe asikwa bese adluliselwa ku 4-ml vial yengilazi levaliwe, bese indzawo yenhloko yavunyelwa kutsi ibutselene kungakapheli emahora lasihlanu. Ngesikhatsi sekukala, i-vial ngayinye beyihlantwa ngemfudlana wemalitha lamabili/lihora lemoya lohlantekile imizuzu lengu 8, lebeyidlule ngaphambilini nge catalyst leniketwa yi Sensor Sense kususa iCO2 kanye nemanti.
Idatha ye-microarray yashicilelwa ekucaleni ku (35) futsi yagcinwa eSikhungweni Savelonkhe Selwatiso Lwetebucwepheshe Betilwane (NCBI) Idathabase Lephelele Yekuveta Emajini (inombolo yekungena GSE30287). Lemininingwane lehambisana nemacembe labangelwa kwelashwa kwe W + OSMs kanye nekulawula lokungalimali kwakhishwa kulolucwaningo. Kucina lokuluhlata ngu log2. Ngaphambi kwekuhlatiya kwetibalo, umgca wesisekelo waguculwa futsi wajwayelekile waba ngemaphesenti awo lange-75 kusetjentiswa iphakheji yesofthiwe ye-R.
Lemininingwane yekucala yekulandzelana kwe RNA (RNA-seq) yetinhlobo te Nicotiana yatsatfwa ku NCBI Ema Archives Ekufundza Lokufishane (SRA), inombolo yephrojekthi yi PRJNA301787, leyabikwa ngu Zhou et al. (39) bese uyachubeka njengobe kuchaziwe ku (56). Lemininingwane leluhlata lecubungulwe yi W + W, W + OSM kanye na W + OSS1 lehambisana netinhlobo te Nicotiana yakhetfwa kutsi ihlaziywe kulolucwaningo, futsi yacubungulwa ngalendlela lelandzelako: Kwekucala, kufundvwa kwe RNA-seq lokuluhlata kwaguculwa kwaba yindlela ye FASTQ. I-HISAT2 igucula i-FASTQ ibe yi-SAM, futsi i-SAMtools igucula emafayela e-SAM abe ngemafayela e-BAM lahlungiwe. StringTie isetjentiselwa kubala kuveta ufuto, futsi indlela yayo yekuveta kutsi kunetincenye ngetincenye letiyinkhulungwane tesisekelo ngetigidzi tetincenye tekubhala letilandzelanako.
Lekholomu ye Acclaim chromatographic (150 mm x 2.1 mm; bukhulu betinhlayiya 2.2μm) lesetjentiswa ekuhlaziyeni kanye nekholomu yekugadza lengu 4 mm x 4 mm ifaka tintfo letifanako. Lendlela lelandzelako ye-binary isetjentiswa eluhlelweni lwe-Dionex UltiMate 3000 Ultra High Liquid Chromatography (UHPLC): 0 kuya ku-0.5 imizuzu, isocratic 90% A [emanti lakhiwe nge-ion, 0.1% (v/v) i-acetonitrile kanye ne-B/0% 0.05% we-esidi ye-formiki); 0.5 kuya ku 23.5 imizuzu, sigaba se gradient ngu 10% A na 90% B, ngekulandzelana; 23.5 kuya ku 25 imizuzu, isocratic 10% A kanye na 90% B. Lizinga lekugeleza lingu 400μl/min. Kuko konkhe kuhlatiya kwe-MS, jova i-eluent yekholomu ibe yi-quadrupole kanye nesikhatsi-sekundiza (qTOF) sekuhlatiya lesifakwe umtfombo we-electrospray losebenta ngendlela ye-ionization lenhle (ivoltheji ye-capillary, 4500 V; umtfombo we-capillary 130 V ; Lizinga lekushisa lekugeleta kwekomisa 200°C;
Yenta kuhlatiya kwetincenye te-MS / MS (lapha kubitwa ngekutsi yi-MS / MS) lengakabaluleki noma lengahlukaniswa nedatha kutfola lwati lwesakhiwo mayelana nephrofayli yekugaya kudla lebonakalako jikelele. Umcondvo wendlela ye-MS / MS lengakhetsi uncike ekutseni i-quadrupole inefasitela lelikhulu kakhulu lekuhlukaniswa kwebuningi [ngako-ke, cabanga ngato tonkhe timphawu te-mass-to-charge ratio (m / z) njengetincenye]. Ngalesizatfu, ngoba lithuluzi le-Impact II alizange likhone kudala i-CE tilt, kuhlatiya lokunyenti lokutimele kwentiwa kusetjentiswa emanani emandla ekushayisana (CE) lakhuphukile labangelwa kushayisana. Ngamafuphi, cala kuhlatiya isampula nge-UHPLC-electrospray ionization / qTOF-MS usebentisa imodi ye-mass spectrometry yinye (timo letiphansi tekuhlukana letikhicitwa kuhlukana emtfonjeni), kuskena kusuka ku-m / z 50 kuya ku-1500 ngemvamisa yekuphindzaphindza ye-5 Hz. Sebentisa initrogen njengegesi yekushayisana yekuhlatiya iMS/MS, bese wenta tilinganiso letitimele kulamavolthi lamane lehlukene lalandzelako ekushayisana-labangelwa kuhlukana: 20, 30, 40, kanye na 50 eV. Kuyo yonkhe inchubo yekukala, i-quadrupole inefasitela lelikhulu lekuhlukaniswa kwebuningi, kusuka ku m/z 50 kuya ku 1500. Uma umtimba wangaphambilini m/z kanye nekuhlolwa kwebubanti bekuhlukaniswa kubekwa ku 200, libanga lebuningi liyasebenta ngekuzenzakalela ngesoftware yekusebenta yetintfo tekusebenta kanye na 0 Da. Skena tincetu te-mass njengasemodini ye-mass yinye. Sebentisa i-sodium formate (50 ml we-isopropanol, 200 μl we-fomic esidi kanye ne-1 ml ye-1M NaOH yesisombululo lesinemanti) kwentela kulinganisa buningi. Kusebentisa i-algorithm yekulinganisa lesetulu yaBruker, ifayela lemininingwane liyalinganiswa ngemuva kwekugijima kwe-spectrum lesemkhatsini ngesikhatsi lesitsite. Sebentisa umsebenti wekutfumela ngaphandle kwesofthiwe yekuhlatiya imininingwane v4.0 (Brook Dalton, eBremen, eJalimane) kugucula emafayela emininingwane leluhlata abe yifomethi ye-NetCDF. Isethi yemininingwane ye-MS/MS igcinwe ku-database levulekile ye-MetaboLights (www.ebi.ac.uk) lenenombolo yekungena. i-MTBLS1471.
MS / MS umhlangano kungafezeka ngekuhlatiya correlation emkhatsini MS1 kanye MS / MS timphawu ikhwalithi for phansi kanye ephezulu ukushayisana amandla kanye nemitsetfo emusha kuphunyeleliswe. Umbhalo we-R usetjentiselwa kucaphela kuhlatiya kwekuhambisana kwekusabalalisa kwe-precursor kumkhicito, kanye nembhalo we-C # (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) usetjentiselwa kusebentisa imitsetfo.
Kute kuncishiswe emaphutsa lamahle langemanga labangelwa ngumsindvo wangemuva kanye nekuhambisana kwemanga lokubangelwa kutfola timphawu letitsite te-m / z kumasampula lambalwa kuphela, sisebentisa umsebenti we “sicoco lesigcwalisiwe” wephakheji ye-R XCMS (yekulungisa umsindvo wangemuva) Kufanele isetjentiswe esikhundleni se “NA peteuak” (peteuak”). Lapho umsebenti wekugcwalisa wesicoco usetjentiswa, kusekhona emanani lamanyenti “0 ′′ emandla kusethi yedatha letawutsintsa kubalwa kwekuhambisana. Ngemuva kwaloko, sicatsanisa imiphumela yekucubungula idatha letfolakala lapho umsebenti wesicoco lesigcwalisiwe usetjentiswa khona futsi lapho umsebenti wesicoco lesigcwalisiwe ungasetjentiswa, bese sibala linani lemsindvo lelingemuva lelilungisiwe lelisekelwe kuleti. emanani emandla lanenani lemuva lelibaliwe Saphindze sacabanga kuphela ngetimphawu letinemandla langetulu kwelinani lelingemuva leliphindvwe katsatfu futsi satitsatsa njenge “ticoco teliciniso.” Kubalwa kwe PCC, kuphela timphawu te m/z te precursor yesampula (MS1) kanye nemasethi emininingwane yetincenye letineticoco letisiphohlongo teliciniso leticatjangwako.
Uma ngabe kucina kwesici selizinga le-precursor kuyo yonkhe isampula kuhambisana kakhulu nekuncipha kwemandla esici selizinga lelifanako lelibhekene nemandla ekushayisana laphansi noma laphakeme, futsi lesici asibhalwa njengesicoco se-isotope yi-CAMERA, singachazwa kabanti. Ngemuva kwaloko, ngekubala bonkhe lababili labakhonakalako be-precursor-product ngaphakatsi kwe-3 s (lifasitelo lesikhatsi sekugcinwa lesilinganiselwe sekugcinwa kwesicoco), kuhlatiya kwekuhambisana kwentiwa. Kuphela uma linani le m/z lingaphansi kwelinani le precursor kanye nekuhlukaniswa kwe MS/MS kwenteka endzaweni lefanako yesampula kusethi yedatha njenge precursor lapho isuselwa khona, kutsatfwa njengesicucu.
Kususelwa kulemitsetfo lemibili lelula, sikhipha tincetu letichaziwe letinemanani e-m/z lamakhulu kune-m/z ye-precursor lekhonjiwe, futsi ngekusebentisa sikhundla sesampula lapho khona i-precursor ivela khona kanye nesicucu lesicacisiwe. Kungenteka futsi kutsi ukhetse timphawu telizinga letikhicitwa tincetu letinyenti letisemtfonjeni letikhicitwa ngendlela ye-MS1 njengetindlela tekucala tebakhetsi, ngaleyo ndlela kukhicite emakhompawundi e-MS / MS langadzingeki. Kute kuncishiswe lokuphindzaphindza kwemininingwane, uma ngabe kufana kwe-NDP kwe-spectra kwedlula 0.6, futsi kungekwe-chromatogram “pcgroup” lebhalwe nge-CAMERA, sitawuhlanganisa. Ekugcineni, sihlanganisa yonkhe imiphumela lemine ye-CE lehambisana ne-precursor kanye netincenye tibe yi-spectrum lehlanganisiwe yekugcina ye-deconvoluted ngekukhetsa sicoco lesiphakeme semandla emkhatsini wato tonkhe ticoco te-candidate letinenani lelifanako le-m / z emandla lehlukene ekushayisana. Tinyatselo tekucubungula letilandzelako tisuselwa kumcondvo we spectrum lehlanganisiwe futsi ticabanga ngetimo letehlukene te CE letidzingekako kwentela kukhulisa ematfuba ekuhlukana, ngoba letinye tincenye tingatfolakala kuphela ngaphansi kwemandla lakhetsekile ekushayisana.
RDPI (30) yasetjentiswa kubala inducibility iphrofayli ukugaya. Lokuhlukahluka kwe-metabolic spectrum (inkomba ye-Hj) kususelwa ebuningi be-MS / MS precursors kusetjentiswa i-Shannon entropy yekusakateka kwemvamisa ye-MS / MS kusetjentiswa le-equation lelandzelako lechazwe nguMartínez et al. (8). Hj = −∑i = 1mPijlog2 (Pij) lapho Pij ihambisana nemvamisa lehlobene ye-MS / MS ye-i-th kusampula ye-j (j = 1, 2, ..., m) (ngi = 1, 2, ..., m) t).
Kucaciswa kwekugaya kudla (Si index) kuchazwa njengekuveta bunjalo be-MS/MS leniketiwe mayelana nemvamisa emkhatsini wemasampula lacatjangwako. Kucaciswa kwe-MS / MS kubalwa njenge-Si = 1t (∑j = 1tPijPilog2PijPi)
Sebentisa lefomula lelandzelako kukala inkomba ye-δj lekhetsekile ye-metabolome yesampula ngayinye ye-j, kanye nesilinganiso se-MS / MS lekhetsekile δj = ∑i = 1mPijSi .
MS/MS spectra kuhambisana ngababili, futsi kufana kubalwa ngekusebentisa emaphuzu lamabili. Kwekucala, kusebentisa i-NDP lejwayelekile (lephindze yatiwe ngekutsi yindlela yekuhambisana ye-cosine), sebentisa lesilinganiso lesilandzelako kute utfole emaphuzu ekufana kwesigaba emkhatsini we-spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 lapho S1 na-S2 kahle njenge-spectrum, 1.1. i imelela sisindvo lesisekelwe emandla esicongweni kutsi umehluko wesicoco lesivamile se-i-th emkhatsini wema spectra lamabili ungaphansi kwa 0.01 Da. Sisindvo sibalwa ngalendlela lelandzelako: W = [kucinisa kwesicoco] m [lizinga] n, m = 0.5, n = 2, njengoba kuphakanyiswe yi MassBank.
Indlela yesibili yekushaya emaphuzu yasetjentiswa, lebeyifaka ekhatsi kuhlatiya i-NL leyabelwana ngayo emkhatsini we-MS / MS. Kute kwenteke loku, sasebentisa tinhlu te-52 NL letivame kuhlangana nato ngesikhatsi senkambo yekuhlukaniswa kwe-MS ngekuhambisana, kanye ne-NL lecondzile kakhulu (ifayela lemininingwane S1) lebeyibhalwe ngaphambilini ye-MS / MS spectrum ye-metabolites yesibili yetinhlobo te-Nepenthes letibutsakatsaka ( 9, 26). Dala i-vector kanambambili ye-1 na-0 ye-MS/MS ngayinye, lehambisana ne-NL lekhona nyalo nalengakho ngekulandzelana. Kususelwa ekufaneni kwebanga le-Euclidean, emaphuzu ekufana kwe-NL abalwa ku-pair ngayinye yema-vectors e-NL kanambambili.
Kute sente kuhlanganiswa lokubili, sasebentisa iphakheji ye-R DiffCoEx, lesekelwe ekwandzisweni kwekuhlatiya kwekuveta ngekubambisana kwe-Gene Lesisindvo (WGCNA). Kusebentisa ema-matrix e-NDP kanye ne-NL yekushaya emaphuzu e-MS / MS spectra, sasebentisa i-DiffCoEx kubala i-matrix yekuhambisana yekucatsanisa. Kuhlanganiswa kwetintfo letimbili kwentiwa ngekubeka ipharamitha ye “cutreeDynamic” kundlela = “hybrid”, cutHeight = 0.9999, deepSplit = T, kanye ne-minClusterSize = 10. Ikhodi yemtfombo we-R ye-DiffCoEx yalandvwa kusuka kufayela lelengetiwe 1 nguTesson et al. (57); Iphakheji yesoftware ledzingekako ye-R WGCNA ingatfolakala ku-https.
Kute sente kuhlatiya inethiwekhi ye-molecule ye-MS / MS, sabala kuxhumana kwe-spectral lokubhanjiwe lokusekelwe etinhlobeni tekufana kwe-NDP ne-NL, futsi sasebentisa isoftware ye-Cytoscape kute sibone ngemehlo engcondvo i-topology yenethiwekhi sisebentisa indlela yekuphila ku-CyFilescape yFiles indlela yekwandzisa i-algorithm.
Sebentisa R version 3.0.1 kwenta kuhlatiya kwetibalo kudatha. Kubaluleka kwetibalo kwahlolwa kusetjentiswa kuhlatiya kwetindlela letimbili tekuhluka (ANOVA), kulandzelwe umehluko lobalulekile ngekwetsembeka (HSD) waTukey wekuhlolwa kwe-post-hoc. Kute kuhlaziywe umehluko emkhatsini wekwelashwa kwetilwane letidla titjalo kanye nekulawula, kusabalalisa lokunemisila lemibili kwemacembu lamabili emasampula ngekuhluka lokufanako kwahlaziywa kusetjentiswa luhlolo lwe Student’s t.
Kute utfole tintfo letengetiwe talesihloko, sicela ubuke http://advances.sciencemag.org/cgi.
Lena yindzatjana levulekile lesakatwa ngaphasi kwemigomo ye-Creative Commons Attribution-Non-Commercial License, levumela kusetjentiswa, kusabalaliswa kanye nekukhicita kabusha nganoma nguyiphi indlela, uma nje kusetjentiswa kwekugcina akusiko kwenzuzo yekutsengisa futsi umcondvo kutsi umsebenti wekucala ulungile. Kudlilisela.
Caphela: Sicela kuphela kutsi unikete likheli lakho le-imeyili kute umuntfu lomncoma kulelikhasi ati kutsi ufuna ayibone le-imeyili nekutsi akusiyo i-spam. Angeke sitsatse noma ngumaphi emakheli e-imeyili.
Lombuto usetjentiselwa kuhlola kutsi usivakashi yini kanye nekuvikela kuhanjiswa kwe-spam lokuzenzakalelayo.
Ithiyori yelwati iniketa imali yemhlaba wonkhe yekucatsanisa ema-metabolomes lakhetsekile kanye nekubiketela kwetifundvo tekuvikela tekuhlola.
Ithiyori yelwati iniketa imali yemhlaba wonkhe yekucatsanisa ema-metabolomes lakhetsekile kanye nekubiketela kwetifundvo tekuvikela tekuhlola.
©2021 Inhlangano Yekutfutfukisa Isayensi YaseMelika. onkhe emalungelo agcinwe. I-AAAS ngumlingani we-HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef kanye ne-COUNTER. Intfutfuko Yesayensi ISSN 2375-2548.