RNA-Seq Raws li De Novo Transcriptome Assembly Thiab Gene Discovery Of Cistanche Deserticola Fleshy Stem-Ⅱ

Functional Classification Ntawm Txhua Cov Ntawv Qhia Tshaj Tawm Raws li Gene Ontology Thiab KEGG Databases

Gene Ontology (GO) annotation tau txais los ntawm UniProt cov lus piav qhia thiab cov ntaub ntawv sib koom ua ke. Nyob rau hauv tag nrho, 20,907 transcripts, accounting rau 32.69% ntawm tag nrho cov qhia sequences, tau muab rau 1,745 cov nqe lus ua haujlwm. Ntawm tag nrho cov ntsiab lus GO ua haujlwm, kev ua haujlwm rau cov txheej txheem lom neeg ua rau feem ntau (1,116, 63.95%) ua raws li cov khoom siv ntawm tes (329, 18.85%) thiab molecular muaj nuj nqi (300, 17.20%). Cov haujlwm uas tau teev tseg ntawm cov ntawv sau tseg tau hais txog ntau yam ntawm GO pawg, thiab 10 GO cov ntsiab lus saum toj kawg nkaus nrog cov ntawv sau tseg tshaj plaws tau teev nyob rau hauv Table 3. Peb muab tag nrho cov ntaub ntawv qhia tawm hauv peb pawg Gene Ontology (Molecular Function, Cellular Cheebtsam, thiab Cov txheej txheem lom neeg) hauv cov ntaub ntawv ntxiv (S3 Dataset). GO cov ntsiab lus hais txog kev sib khi ua haujlwm thiab kev ua haujlwm hloov pauv tau feem ntau sawv cev hauv pawg molecular muaj nuj nqi. Hais txog kev sib khi ua haujlwm, cation binding (4,394 daim ntawv teev lus) sawv cev rau ntau tshaj, ua raws li nucleotide / nucleoside binding (3,404 cov ntaub ntawv nyob rau nruab nrab) thiab protein khi (2,422 cov ntawv sau). Thaum nyob hauv pawg neeg ua haujlwm hloov pauv, feem ntau yog cov uas hloov cov phosphorus-muaj pawg (2,256 cov ntawv sau tseg, 65.77%). Ntawm cov kab ke ntawm tes, cov ntawv sau tau ntau dua nyob rau hauv lub cellular (10,581 cov ntawv sau tseg nyob rau nruab nrab), thaum ntawm cov txheej txheem lom neeg, cov ntaub ntawv sau tseg tau koom nrog ntau dua hauv cov txheej txheem biopolymer metabolic (6,683 cov ntawv sau nruab nrab), ua raws li kev cai ntawm cov txheej txheem cellular (4,841 cov ntawv sau tseg. ), gene qhia (4,678 transcripts) thiab thauj (3,512 transcripts).

NATURAL CISTANCHE TUBULOSA RAU KEV PAB CUAM ALZHEIMER PHGS75% ECH 30% ACT 12%

Rau kuv cov noob koom nrog hauv biosynthesis ntawm lignin thiab PhG, 21,358 qhov tsis muaj peev xwm ua kom muaj protein ntau tau tshawb nrhiav cov noob kab ke ntawm 13 cov kab mob hauv KEGG database, thiab lawv tau muab rau 275 KEGG txoj hauv kev nrog tsawg kawg 5 hits. Sab saum toj 10 txoj hauv kev uas feem ntau ua raws cov kab ke tau teev tseg hauv Table 4. Feem ntau txoj hauv kev tau koom nrog hauv cov txheej txheem metabolic, xws li amino acid lossis protein metabolism (ko01230, ko04141, thiab ko04120), carbohydrate metabolism (ko01200 thiab ko00500), thiab nucleotide los yog nucleoside metabolism (ko03018, ko00230, thiab ko00240). Tsis tas li ntawd, muaj 27 txoj hauv kev ntsig txog cov metabolism hauv nruab nrab (Daim duab 2), xws li terpenoid txha caj qaum biosynthesis, phenylpropanoid biosynthesis, carotenoid biosynthesis, isoquinoline alkaloid biosynthesis, thiab tropane, piperidine thiab pyridine alkaloid biosynthesis. Cov txiaj ntsig no qhia ntxiv qhia tias cov txheej txheem metabolic tseem tab tom ua hauvC. deserticolaqia cov ntaub so ntswg. Tag nrho cov ntaub ntawv qhia txog kev cuam tshuam nrog KEGG txoj hauv kev tau teev nyob rau hauv cov ntaub ntawv ntxiv (S4 Dataset). Txawm hais tias muaj qee qhov kev hloov pauv loj ntawm C. deserticola thiab lwm yam nroj tsuag, xws li mov (S5 Dataset), peb lub hom phiaj tseem ceeb hauv txoj kev tshawb no yog nthuav tawm tag nrho cov ntaub ntawv sau tseg ntawm C. deserticola qia thiab duab txog txoj hauv kev ntawm PhGs biosynthesis. uas yuav pab tau rau kev coj noj coj ua.

Cov neeg sib tw Genes Encoding Enzymes koom nrog hauv Biosynthesis ntawm Lignin

Lignin yog qhov thib ob ntau tshaj plaws nyob rau hauv lub ntiaj teb cov av polymer nyob rau hauv cov nroj tsuag lub nceeg vaj, tsim mus txog rau ib feem peb ntawm cov khoom pom nyob rau hauv cov nroj tsuag cell phab ntsa. Raws li ib qho tseem ceeb ntawm cov phab ntsa ntawm tes, lignins pab kev thauj mus los hauv dej, muab kev txhawb nqa txhua yam thiab kev ncaj ncees, thiab tiv thaiv cov kab mob thiab cov tsiaj txhu. Cov luag haujlwm ntawm lignin muaj txiaj ntsig zoo hauv kev txhawb nqa kev loj hlob hauv av ntawm C. deserticola hauv suab puam. Hauv txoj kev tshawb no, peb nthuav tawm cov duab tiav ntawm lignin biosynthesis txoj hauv kev hauv C. deserticola (Daim duab 3), uas cov lignin monomers yog biosynthesized los ntawm phenylalanine los ntawm ntau cov tshuaj enzymatic, nrog rau hydroxylation, methylation, txo, thiab oxidative polymerization txheej txheem. Lignin biosynthesis-related enzymes raug kuaj pom rau peb hom kev sib xyaw ua ke hauv cov hlab ntsha (p-hydroxyl-phenyl (H), guaiacyl (G) thiab syringyl (S) lignin) thiab 5-hydroxyl-guaiacyl lignin uas tsuas yog txheeb xyuas. nyob rau hauv COMT (caffeic acid 3-O-methyltransferase, EC 2.1.1.68) deficiency (xws li knock-down) cov nroj tsuag.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) yog thawj enzyme tseem ceeb hauv txoj kev lignin biosynthesis (Daim duab 3) uas hloov phenylalanine rau hauv cinnamic acid los ntawm non-oxidative deamination. Tag nrho ntawm 6,297 PAL nyeem tau ua ntu zus thiab 7 PAL cov ntawv sau tseg tau sib sau ua ke hauv C. deserticola (Table 5). Los ntawm kev sib piv qhov sib piv, peb pom tias 4 ntawm lawv (comp28550_c1_seq1/2/3/5) muaj ntau dua 95% zoo sib xws nrog cov paub mRNA sib lawv liag ntawm C. deserticola (gi| 289595227|gb|ADD12041.1|), thaum comp28550_c1_seq4 thiab comp25940_c0_seq1 muaj 77% thiab 82% zoo sib xws, feem. ORF kev twv ua ntej tau nthuav tawm 5 cov ntawv teev lus muaj peev xwm ntawm cov proteins encoding thiab nqa nrog cov tshuaj tsw qab amino acid lyase domain (PF00221.14). Ntawm lawv, tsuas yog cov comp28550_c1_seq4 transcript tuaj yeem ua tiav cov protein ntau ntawm 718 amino acid residues. Nws tau raug tshaj tawm tias PAL tau encoded los ntawm ib tsev neeg me me hauv ntau hom nroj tsuag, xws li 4 hauv Arabidopsis thaliana, 5 hauv Populus trichocarpa, 3 hauv Scutellaria baicalensis, thiab 7 Cucumis sativus, thiab lwm yam. Peb qhov kev soj ntsuam phylogenetic qhia tias muaj 4. PAL-encoding noob hauv C.

deserticola thiab peb muab lawv lub npe CdPAL1, CdPAL2, CdPAL3 thiab CdPAL4, ntsig txog (S2 Fig). 4-coumarate-CoA ligase (4CL, EC 6.2.1.12) thiab trans-cinnamate 4-monooxygenase (CYP73A, EC 1.14.13.11) yog ob lub enzymes lub luag haujlwm rau kev hloov cinnamic acid rau dicoumarol-CoA hauv ob qhov rov qab xaj. Lawv kuj tseem nyob hauv cov pob txha, thiab lawv cov lus qhia FPKM qhov tseem ceeb yog 39.57 thiab 51.93, raws li.

Plaub hom lignins tau biosynthesized los ntawm ntau txoj kev uas tau tswj los ntawm peb lub ntsiab enzymes, cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), shikimate o-hydroxycinnamoyltransferase (HCT, EC 2.3.1.133), thiab ferulate{{1{1}} {56}}}}hydroxylase (F5H, EC 1.14.-.-). CCR tau tshaj tawm tias yog qhov chaw tswj ntawm txoj kev lignins [50, 51] uas catalyzed X-CoA (X suav nrog dicoumarol, caffeoyl, feruloyl, 5-hydroxyl-feruloyl, thiab sinapoyl) rau hauv Y-aldehyde (Y suav nrog p -cougar, caffeoyl, coniferyl, 5-hydroxyl-coniferyl, thiab snap), thaum HCT catalyzed p-coumaroyl-CoA rau p-coumaroyl shikimic acid/p-coumaroyl quinic acid. Ob lub enzymes, ib yam li hloov pauv, tswj cov biosynthesis ntawm P-hydroxyl-phenyl lignins lossis lwm yam peb hom lignins. F5H yog lwm ceg hloov pauv uas tswj syringyl lignin thiab 5-hydroxyl-guaiacyl lignin. Lwm cov enzymes tseem ceeb suav nrog caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.68), caffeoyl-CoA O-methyltransferase (CCoAOMT, EC 2.1.1.104), thiab cinnamyl-alcohol dehydrogenase (CAD, EC 1.5.1.1.1. ) kuj pom tau hais. Cov ncauj lus kom ntxaws qhia tau teev nyob rau hauv Table 6. Cov enzyme genes uas tau txheeb xyuas hauv txoj kev tshawb no yuav muab ib qho txiaj ntsig zoo rau kev ua haujlwm genomic hauv cov nroj tsuag tshuaj tseem ceeb no. 10 cov noob muaj feem xyuam rau txoj kev lignins biosynthesis hauv Table 6 tau raug xaiv rau RT-qPCR pov thawj kom paub meej tias peb cov txiaj ntsig RNAseq (Daim duab 4), thiab lawv cov kev sib raug zoo siab (Pearson correlation coefficient: 0.90343) tau qhia qhov tseeb siab thiab rov ua dua ntawm peb qhov kev txheeb xyuas cov ntaub ntawv. S1 Dataset teev cov txheej txheem primer siv hauv qhov kev tshuaj ntsuam no.

NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev sib deev PHGS75% ECH 30% ACT 12%

Cov neeg sib tw Genes Encoding Enzymes koom nrog hauv Biosynthesis ntawm PhGs

Phenylethanoid glycosides (PhGs) paub tias yog cov khoom xyaw tseem ceeb hauv C. deserticola nrog cov dej num ntawm kev txhim kho kev sib deev, tshem tawm cov dawb radicals, thiab tiv thaiv kev laus. Peb cov tshuaj lom neeg ntawm PhGs yog cov organic acid, saccharide, thiab phenylethanol aglycon (Fig 3). Cov organic acids suav nrog caffeic acid, ferulic acid, thiab coumalic acid yog cov khoom ntawm phenylpropanoid biosynthesis txoj kev. Cov khoom ntawm saccharide nrog rau cov piam thaj thiab rhamnose yog cov khoom ntawm carbohydrate metabolism txoj hauv kev, xws li cov hmoov txhuv nplej siab thiab sucrose metabolism, amino qab zib thiab nucleotide qab zib metabolism, fructose thiab mannose metabolism, thiab lwm yam. Txawm li cas los xij, txoj kev biosynthesis ntawm phenylethanol ib feem tseem tsis tau meej meej. Ntawm no, peb tau npaj ob txoj hauv kev tau phenylethanol biosynthesis raws li peb cov ntaub ntawv ua ntu zus. Ib qho yog txoj kev tshaj tawm caffeic acid lossis ferulic acid txoj hauv kev, tseem hu ua cinnamic acid txoj kev uas zoo ib yam li lignin biosynthesis qaum txoj kev. Lwm qhov yog raws li txoj hauv kev phenylalanine metabolism (Daim duab 3), uas phenylalanine rau phenylethanol tau ua tiav los ntawm kev paub 'Enrlich txoj kev' uas tau pom thawj zaug hauv cov poov xab ib puas xyoo dhau los thiab siv tau hauv petunia paj, txiv lws suav, thiab sawv. Plaub enzymes encoding aspartate / tyrosine aminotransferase, histidine-phosphate aminotransferase, thiab thawj-amine oxidase uas yog lub luag haujlwm rau kev hloov pauv ntawm phenylalanine rau phenylethanol tau kuaj pom nyob rau hauv qia ntawm C. deserticola. Cov khoom ntawm phenyl ethanol tej zaum yuav ntxiv oxidized los ntawm monooxygenase los yog methylated los ntawm methyltransferase rau hauv nws cov derivates (phenyl ethanol aglycon) uas koom nrog hauv PhG biosynthesis. Hauv cov ntsiab lus, ob txoj hauv kev biosynthesis ntawm phenylethanol aglycon tau npaj rauC. deserticolatab sis tseem xav tau kev kawm ntxiv ntxiv.

Kev sib tham

Nyob rau hauv xyoo tas los no, cog genomics tau tsim sai heev nrog rau kev siv cov thev naus laus zis txuas ntxiv mus, thaum qee qhov kev tshawb fawb tau tsom mus rau genomics ntawm cov nroj tsuag tshuaj ntsuab. Nws yog qhov tsim nyog yuav tsum tau ua qhov kev tshawb fawb genomic lossis transcriptomic kom nkag siab txog nws qhov kev hloov pauv rau qhov chaw qhuav thiab dej ntsev thiab txoj hauv kev biosynthesis ntawm cov khoom tseem ceeb bioactive. Lub de novo transcriptome discovery rau qee cov nroj tsuag kho mob,

xws li Panax ginseng, Ginkgo biloba, thiab Glycyrrhiza uralensis tau xub siv los siv Roche 454 platform rau nws qhov ntev nyeem ntawv. Vim tias muaj peev xwm sib dhos ua tau zoo nrog kev nyeem luv luv, tshwj xeeb tshaj yog cov ntawv nyeem tau zoo kawg nkaus, Illumina-based transcriptome sequencing thiab sib dhos kuj tau siv dav rau cov qauv thiab cov kab mob uas tsis yog qauv. Hauv txoj kev tshawb fawb tam sim no, peb tau tsim txog 8G ntawm 101 bp ua khub-kawg nyeem thiab tsim cov unigene txuas ntxiv nrog 725 bp qhov nruab nrab ntev. Cov ntaub ntawv loj-txheej txheem transcriptome tuaj yeem muab cov ntaub ntawv siv tau zoo thiab siv los txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau ua rau cov txiav, cov ntaub ntawv tseem ceeb ntawm cov qia loj tuaj yeem muab cov ntaub ntawv siv tau zoo thiab siv los ua cov txheej txheem metabolic ntawm cov khoom siv bioactive ntawm C. deserticola. Muaj 81.62% ntawm tag nrho cov ntaub ntawv nyoos dhau los ntawm cov ntxaij lim dej nruj (nrog rau kev txiav cov adapter trimming thiab tsis zoo nyeem cov ntawv pov tseg) ua ntej sib dhos, tawm tswv yim zoo ntawm peb cov ntaub ntawv sib txuas, thiab 82.08% ntawm cov ntawv nyeem tau zoo yog qhov tseem ceeb rau kev sib dhos. Lwm cov ntawv nyeem uas tsis tuaj yeem siv rau kev sib dhos tuaj yeem yog los ntawm kev sib txuas ua ke, kev sib dhos tsis zoo li al. Cov ntawv nyeem uas tsis siv tau zoo tseem muaj txiaj ntsig zoo hauv kev txhim kho de novo sib dhos ua ke nrog kev nyeem ntev dua los ntawm lwm lub platform (xws li Roche 454) yav tom ntej.

NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev sib deev PHGS75% ECH 30% ACT 12%

Ntau tus lej ntawm cov ntawv sau sib sau ua ke (30,098) tau pom qhov sib luag zoo sib xws rau cov noob paub hauv cov ntaub ntawv pej xeem, qhia tias peb cov ntaub ntawv Illumina-raws li khub-kawg tau npog ib feem ntau ntawm cov ntawv sau ntawm C. deserticola. Cov ntawv sau tseg uas tsis muaj BLAST hits tej zaum yuav yog vim 3' lossis 5' cov cheeb tsam tsis tau txhais, tsis yog-coding RNA, lossis cov kab ke tshiab ntawm C. deserticola. Cov ntawv teev lus tau hais tseg tau sau tseg rau ntau yam ntawm GO pawg thiab KEGG txoj hauv kev (Tables 3 thiab 4), uas ntau cov ntawv sau tau raug xa mus rau cov txheej txheem ntsig txog cov metabolism hauv theem nrab. Raws li peb paub, phenylpropanoid tuaj yeem ua haujlwm raws li cov tshuaj tiv thaiv kab mob inducible nrog salutary zoo rau kev ua neej nyob hauv av [1], thiab tseem ua raws li lub teeb liab molecule hauv cov nroj tsuag-microbe kev sib cuam tshuam nrog rau nws cov khoom siv tshuaj [68, 69]. Terpenoid yog siv rau biosynthesis ntawm bioactive Cheebtsam (xws li 6- deoxycatalpol) [70]. Peb pom cov noob koom nrog hauv phenylpropanoid thiab terpenoid caj qaum biosynthesis txoj hauv kev muaj ntau heev hauv C. deserticola. Qhov tseem ceeb tshaj, qhov kev tshawb pom ntawm txoj hauv kev zoo ntawm lignin biosynthesis (Daim duab 3) qhia txog cov txheej txheem metabolic ntawm lignin hauv C. deserticola qia. Tag nrho cov enzymes paub txog kev koom tes hauv biosynthesis ntawm lignin (Daim duab 3) tau kuaj pom, thiab plaub lub enzymes tseem ceeb suav nrog PAL, CCR, HCT, thiab F5H muaj qis qhia ntau (FPKM 26.47, 3.89, 3.4, thiab 3.83, raws li) piv nrog Lwm cov enzymes noob (Table 6). Txawm hais tias qhov kev hloov pauv ntawm peb lub noob tuaj yeem cuam tshuam lignin ntau lawm hauv C. deserticola yog qhov tsim nyog rau kev kawm ntxiv. PAL yog ib qho enzyme tseem ceeb hauv lignin biosynthesis thiab tseem koom nrog hauv biosynthesis ntawm phenylpropanoid, resveratrol, flavonoid, thiab coumarin [71-74]. Peb kuaj pom plaub PAL genes nyob rau hauv C. deserticola genome (S2 Fig) uas yog coincident nrog uas PAL tau encoded los ntawm ib tug me me multigene tsev neeg [39, 43, 45–49] thiab ntxiv pov thawj nws yuav ua si lub luag hauj lwm tseem ceeb hauv metabolic carbon flux. .

PhG yog cov khoom xyaw tseem ceeb hauv C. deserticola. Cov noob koom nrog hauv biosynthesis ntawm phenylethanol yog qhov tseem ceeb rau qhov zoo ntawm C. deserticola. Peb tau txiav tawm ob txoj hauv kev sib txawv ntawm biosynthesis ntawm phenyl ethanol thiab 17 enzyme noob koom nrog hauv PhG biosynthesis hauv C. deserticola qia. Qhov ua tau tom qab caffeic / ferulic acid cov txheej txheem (Daim duab 3) kuj raug txiav tawm thawj zaug raws li cov qauv qauv ntawm cov khoom nruab nrab thiab cov khoom catalytic ntawm cov enzymes sib xws, uas cov caffeic / ferulic acid yuav yog thawj zaug oxidized rau phenylpyruvate derivate; tom qab ntawd, pawg carboxyl raug tshem tawm los ntawm decarboxylases; Thaum kawg, pawg aldehyde tau hloov rov qab rau hauv pawg cawv los ntawm dehydrogenase. Qhov no yog thawj daim ntawv thov ntawm Illumina paired-kawg sequencing thev naus laus zis los tshawb xyuas tag nrho cov ntawv sau tseg ntawm C. deserticola thiab sib sau RNA-seq nyeem yam tsis muaj kev siv genome. Txoj kev tshawb no yuav muab cov peev txheej muaj txiaj ntsig thiab cov txheej txheem noob rau kev ua haujlwm genomics thiab kev tshawb fawb proteomics ntawm C. deserticola yav tom ntej.

Cov lus xaus

Hauv txoj kev tshawb no, peb tau sau cov ntaub ntawv sau tseg ntawm C. deserticola qia raws li cov ntaub ntawv sib luag ntawm cov ntaub ntawv siab, txheeb xyuas cov noob koom nrog hauv biosynthesis txoj hauv kev ntawm lignin, thiab tseem suav tias muaj peev xwm biosynthesis txoj hauv kev ntawm PhGs thawj zaug, uas yuav ua rau kom nkag siab. ntawm cov txheej txheem physiological tsis meej thiab cov tshuaj muaj txiaj ntsig zoo hauv qib molecular. Txog rau tam sim no, qhov no yog thawj zaug sim de novo sib sau ua ke tag nrho cov ntawv sau tseg ntawm C. deserticola qia thiab txhawm rau txheeb xyuas txoj hauv kev biosynthesis ntawm cov tshuaj siv tshuaj siv Illumina-based sequencing datasets. Peb txoj kev tshawb fawb tuaj yeem txhawb txoj kev loj hlob ntawm cov tshuaj ntuj thiab xaiv cov cultivars nrog cov yam ntxwv ntawm cov tshuaj.

NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev sib deev PHGS75% ECH 30% ACT 12%