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

Keeb kwm

Cistanche deserticola yog ib tsob nroj uas tsis muaj yees duab tua kab mob uas muaj txiaj ntsig zoo thiab feem ntau faib rau hauv cov suab puam ntawm Northwest Tuam Tshoj. Nws qhuav fleshy qia yog ib qho tseem ceeb tonic nyob rau hauvtshuaj suav tshuajnrog rau lub luag haujlwm ntawm kev txhim kho txiv neej kev ua haujlwm ntawm kev sib deev thiab txhawb kev tiv thaiv kab mob, tab sis ob peb txoj kev tshawb fawb txog kev siv tshuab tau ua ib feem vim tsis muaj cov khoom siv genomic thiab transcriptomic.

NATURAL CISTANCHE TUBULOSA CHINESE TRADITIONAL MEDICINE PHGS75% ECH 30% ACT 12%

Cov txiaj ntsig

Nyob rau hauv txoj kev tshawb no, peb tau ua qhov sib sib zog nqus transcriptome sequencing nyob rau hauv lub cev nqaij daim tawv C. deserticola, thiab hais txog 80 lab nyeem tau tsim siv Illumina khub-kawg sequencing ntawm HiSeq2000 platform. Siv lub trinity assembler, peb tau txais 95,787 transcript sequences nrog transcript lengths xws li 200bp rau 15,698bp, muaj qhov nruab nrab ntev ntawm 950 bases thiab N50 ntev ntawm 1,519 bases. 63,957 transcripts tau raug txheeb xyuas raws li nquag qhia nrog FPKM Ntau dua lossis sib npaug rau 0.5, uas 30,098 cov ntawv sau tseg tau sau tseg nrog cov lus piav qhia gene lossis cov ntsiab lus ntawm cov noob caj noob ces los ntawm kev soj ntsuam zoo sib xws rau ntau cov ntaub ntawv pej xeem (Uniprot, NR, thiab Nt ntawm NC) . Tsis tas li ntawd, peb tau txheeb xyuas cov noob caj noob ces tseem ceeb hauv biosynthesis ntawm lignin thiab phenylethanoid glycosides (PhGs) uas paub tias yog cov khoom xyaw tseem ceeb. Plaub phenylalanine ammonia-lyase (PAL) noob, thawj enzyme tseem ceeb hauv lignin thiab PhG biosynthesis raug txheeb xyuas raws li kev sib piv sib piv thiab kev soj ntsuam phylogenetic. Ob txoj hauv kev biosynthesis ntawm PhGs kuj tau thov thawj zaug.

Cov lus xaus

Nyob rau hauv tag nrho, peb ua tiav ib qho kev ntsuam xyuas thoob ntiaj teb ntawm C. deserticola fleshy qia transcriptome siv RNA-seq thev naus laus zis. Ib qho kev sau ntawm cov noob caj noob ces muaj feem xyuam rau biosynthesis ntawm lignin thiab phenylethanoid glycosides tau txheeb xyuas los ntawm cov ntawv sau ua ke thiab sau tseg, thiab cov noob tsev neeg ntawm PAL kuj tau kwv yees. Cov ntaub ntawv ua ntu zus los ntawm txoj kev tshawb fawb no yuav muab cov peev txheej tseem ceeb rau kev tshawb fawb yav tom ntej phenylethanoid glycosides biosynthesis thiab kev ua haujlwm genomic hauv cov nroj tsuag tshuaj tseem ceeb no.

Taw qhia

C. deserticola yog ib tug thoob ntiaj teb genus ntawm perennial suab puam nroj tsuag los ntawm Orobanchaceae tsev neeg thiab yog ib tug kiag li tsis-photosynthetic hom thiab feem ntau loj hlob underground holoparasitic nroj tsuag. Nws yog parasitized ntawm cov hauv paus hniav ntawm psammophyte Haloxylon ammodendron (Chenopodiaceae), uas feem ntau nyob hauv cov suab puam thiab cov suab puam ib nrab vim nws ua siab ntev rau drought thiab salinity. C. deserticola qhia tau hais tias muaj zog tiv thaiv ib puag ncig hnyav thiab feem ntau yog faib rau Northwest Tuam Tshoj, tshwj xeeb tshaj yog nyob rau sab hauv Mongolia, Gansu, thiab Xinjiang. Nws raug suav hais tias yog hom tsiaj qus uas muaj kev puas tsuaj nyob rau xyoo tas los no vim muaj kev noj ntau los ntawm tib neeg. C. deserticola uas feem ntau hu ua suab puam ginseng feem ntau hu ua suab puam-broomrape thiab cov qia qhuav tau siv los ua ib qho tseem ceeb tonic hauv Suav teb thiab Nyiv tau ntau xyoo. Nws tau pib sau tseg hauv Shen Nong Ben Cao Jing (Cov Lus Txhais ntawm Suav Materia Medica, 1977) kwv yees li 1800 xyoo dhau los thiab tau suav tias yog ib qho ntawm cov ntsiab lus tseem ceeb ntawmSuav tshuaj ntsuab Cistanche.

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

Cov extracts ntawm C. deserticola muaj ntau yam tshuaj ua haujlwm, tshwj xeeb tshaj yog siv los txhim kho kev ua haujlwm ntawm kev sib deev, tonifying ob lub raum, tiv thaiv daim siab, kev ua haujlwm aperient, txhim kho kev nco, immunomodulatory, kev ua haujlwm antioxidative, anti-inflammatory, antiviral kev ua, thiab lwm yam. Qhov tseem ceeb bioactive Cheebtsam ntawm C. deserticola yog Phenylethanoid glycosides (PheGs, PhGs). Txog rau tam sim no, ntau tshaj 20 phenylethanoid glycosides tau raug cais tawm ntawm cov qia succulent ntawm C.deserticola. Ntawm lawv,acteoside thiab echinacosideyog ob lub ntsiab lus tseem ceeb nrog cov haujlwm tseem ceeb ntawm cov tshuaj thiab tau sau tseg raws li cov qauv zoo ntawm C. deserticola hauv Suav pharmacopeia (2005 thiab 2010 tsab). Peb cov tshuaj lom neeg ntawm PhGs yog cov organic acid, saccharide, thiab phenylethanoid, txawm li cas los xij, cov ntsiab lus hais txog phenylethanoid biosynthetic pathways tseem tsis to taub hauv C.deserticola.

Txawm hais tias kev lag luam thiab tshuaj tseem ceeb ntawm C.deserticola, cov ntaub ntawv genomic thiab transcriptomic ntawm hom no muaj tsawg heev. Tsis muaj ESTs muaj nyob rau hauv NCBI database thiab tag nrho cov ntaub ntawv genome rau hom no tseem tsis muaj tshwj tsis yog rau chloroplast genome ib ntus. Cov ntaub ntawv transcriptomic txwv tsis pub cuam tshuam txoj kev tshawb fawb ntawm PhG biosynthetic mechanisms. RNA-seq thev naus laus zis tuaj yeem tsim cov kab ke ntawm cov lus qhia ntawm lub hom phiaj genome thiab txheeb xyuas cov noob [18] siv NGS thev naus laus zis platforms (xws li Applied Biosystems SOLiD, Illumina HiSeq, thiab Roche 454). Nws tau dhau los ua neeg nyiam nyob rau hauv kev sib sau ua ke ntawm novo, vim nws yog tus nqi-zoo thiab muaj zog mus kom ze nrog kev daws teeb meem siab thiab dav dav, tshwj xeeb tshaj yog vim nws muaj txiaj ntsig los tshawb nrhiav cov ntawv sau qis. Vim muaj ntau yam zoo, RNA-seq tshwj xeeb tshaj yog txaus nyiam rau cov kab mob uas tsis yog qauv nrog cov peev txheej caj ces tsawg. Txawm li cas los xij, tsis muaj kev tshawb fawb ntxaws txog C. deserticola transcriptome los ntawm RNA-seq.

Nyob rau hauv txoj kev tshawb no, peb thoob plaws ntiaj teb tau ua raws li cov ntawv sau cia rau C. deserticola siv Illumina Hiseq2000 platform thiab tau txais 7.9G raw cov ntaub ntawv. Los ntawm kev sib sau ua ke thiab cov lus piav qhia, peb mined cov noob koom nrog hauv biosynthesis ntawm PhG thiab cov noob muaj lub luag haujlwm rau tag nrho lignin biosynthesis. Peb qhov kev soj ntsuam RNA-seq tau tsim thawj C. deserticola kev pom zoo transcriptome thiab muab kev nkag siab tshiab rau hauv kev nkag siab zoo ntawm cov nqi tshuaj ntawm C. deserticola. Tsis tas li ntawd, cov txheej txheem tau piav qhia ntawm no tuaj yeem siv dav rau cov ntaub ntawv sau npe los pab txhawb kev tshawb pom cov noob koom nrog hauv cov tshuaj lom neeg tshwj xeeb biosynthesis txoj hauv kev hauv lwm cov nroj tsuag tshuaj uas muaj kev txwv genomic heev.

Cov ntaub ntawv thiab cov txheej txheem

Cov khoom siv cog qoob loo

Lub qia tshiab succulent rau C. deserticola nyob rau hauv lub excavation theem tau sau los ntawm ib tug cog hauv paus nyob rau hauv BayanHot City ntawm Alxa League nyob rau hauv Inner Mongolia nyob rau sab qaum teb Tuam Tshoj. Daim ntawv tso cai sau tau los ntawm tus tswv (HongKui CongRong Group) ntawm lub hauv paus cog. Daim ntawv pov thawj tau muab tso rau hauv Core Genomic Facility ntawm Beijing Institute of Genomics, Suav Academy ntawm Sciences. Tom qab ntxuav, cov succulent qia cov ntaub so ntswg raug txiav mus rau hauv me me pieces thiab tam sim ntawd khov nyob rau hauv cov kua nitrogen, thiab ces muab cia rau ntawm -80 degree kom txog rau thaum ua ntxiv.

RNA rho tawm, cDNA tsev qiv ntawv tsim, thiab Illumina sequencing

Tag Nrho RNA tau muab rho tawm los ntawm cov qia succulent siv TRIzol Reagent (Invitrogen Inc., California, USA) raws li cov chaw tsim khoom cov lus qhia. Cov qauv ua tau raug kho nrog DNase I kom tshem tawm cov genomic DNA. Extracted RNAs raug suav hais tias siv Agilent 2100 bioanalyzer (Agilent Technologies) thiab kuaj xyuas kev ncaj ncees siv denaturing agarose gel electrophoresis nrog ethidium bromide staining. Cov qauv RNA nrog A260 / A280 qhov sib piv ntawm 1.9 thiab 2.1, RNA 28S: 18S qhov sib piv siab dua 1.0, thiab RNA tus lej kev ncaj ncees (RINs) -8.5 tau siv hauv kev tshuaj ntsuam tom ntej.

Cov tsev qiv ntawv RNA-seq tau tsim los siv Illumina Truseq RNA Cov Khoom Siv Npaj Ua Ntej. Poly(A) + RNA raug cais tawm ntawm tag nrho RNA siv Dynal ligo(dT)25 hlaws raws li cov chaw tsim khoom cov lus qhia. Tom qab purification, ib qho kev sib cais tsis tau ntxiv los rhuav tshem mRNA rau hauv cov khoom luv luv. Thawj-strand cDNA tau tsim los siv cov khoom luv luv no ua cov qauv, nrog rau SuperScript III thim rov qab transcriptase thiab N6 random hexamer primer. Thib ob-strand cDNA tom qab ntawd tau tsim los siv cov tsis, dNTPs, RNaseH, thiab DNA polymerase I. Qhov tshwm sim ob-stranded cDNA tau raug kho kawg siv T4 DNA polymerase, DNA polymerase I Klenow fragment, thiab T4 polynucleotide kinase, thiab ligated rau adapters siv T4 DNA ligase. Adaptor-ligated fragments tau huv si siv QiaQuick PCR cov khoom siv rho tawm thiab eluted nrog EB tsis. Tom qab kev tshuaj xyuas siv agarose gel electrophoresis, cov khoom tsim nyog raug xaiv los ua cov qauv rau PCR amplification. Sequencing ntawm lub tsev qiv ntawv cDNA tau ua nrog Illumina HiSeq 2000 system.

Transcripts de novo los ua ke thiab gene qhia kom muaj nuj nqis

Raw nyeem generated los ntawm sequencing raug ntxuav los ntawm kev tshem tawm lub adapter sequences (ATCTCGTATGCCGTC) siv ib tug hauv tsev txoj kev. Tom qab ntawd peb tau ua cov txheej txheem lim dej tsis zoo. Ua ntej, cov hauv paus nrog phred zoo qhab nia qis dua 20 yuav raug txiav los ntawm 3' kawg ntawm qhov sib lawv liag, kom txog rau thaum khiav mus rau hauv ib lub hauv paus nrog qhov zoo dua (Ntau dua lossis sib npaug rau 20). Yog tias qhov ntev nyeem ntawv luv dua 50bp, nws yuav raug muab pov tseg. Qhov thib ob, kev nyeem ntawv yuav raug lim ntxiv los ntawm cov txheej txheem uas 70% ntawm cov hauv paus hauv ib qho kev nyeem muaj cov qhab nia zoo (Ntau dua lossis sib npaug rau 20). Thirdly, tsuas yog khub-kawg nyeem tau siv rau kev sib dhos ntxiv. De novo transcript sib dhos tau ua los ntawm Trinity tso tawm _20130216 [30] uas muaj peb lub software txuas ntxiv: Inchworm, Chrysalis, thiab Npauj Npaim. Cov txheej txheem sib dhos tau teeb tsa raws li hauv qab no:-seqType fq-JM 300G -min_contig_ntev 200-CPU 20-inchworm_cpu {{21} }bflyCPU 20.

Txhawm rau kom muaj nuj nqis ntawm cov ntawv sau ntau, cov ntawv sib txuas ua ke-kawg nyeem tau rov ua raws li cov ntawv sau ua ke uas siv cov ntawv sau hauv Trinity. Cov ntawv nyeem tau siv rau kev ntsuas los ntawm RSEM (RNA-Seq los ntawm Kev Cia Siab Maximization) software. Gene los yog isoform abundance tau sawv cev los ntawm cov fragment per kilobase ntawm transcript ib lab fragment mapped (FPKM) tus nqi, cov transcripts nrog FPKM tus nqi sib npaug los yog loj dua 0.05 tau txhais raws li qhia.

Cov lus piav qhia ua haujlwm ntawm cov ntawv sau tseg

Tsis muaj gene annotation teev ntawm C. deserticola tsuas yog rau chloroplast genome [1]. Peb tau piav qhia cov ntawv sau tseg los ntawm kev muab piv rau Genbank Nt, Genbank Nr, thiab TAIR10_ pep_20101214_ hloov kho cov ntaub ntawv sib cais siv BLAST program (E< = 1e-20). Meanwhile, all expressed transcripts were translated into potential proteins according to ORF prediction by TransDecoder and predicated for the conserved domains based on the Pfam database.

Gene Ontology thiab KEGG txoj hauv kev annotation Los ntawm kev sib txuas ua ke zoo sib xws rau Uniprot database (Gene Ontology (GO) annotation ntawm tag nrho cov ntawv sau ua ke tau txais los ntawm kev siv lub koom haum cov ntaub ntawv rub tawm los ntawm (ftp://ftp.ebi.ac.uk/pub/ databases/GO/goa/UNIPROT/gene_sociation goa_uniprot.gz). CC, BP, thiab MF pawg sib cais.

KEGG cov ntaub ntawv taug kev tau raug muab rau txhua qhov kev kwv yees cov protein uas siv cov cuab yeej online KAAS (KEGG Automatic Annotation Server) [34]. Cov kab ke hauv fasta tau xa mus rau KAAS thov, thiab cov ntaub ntawv tshwm sim ntawm txhua txoj hauv kev cov ntaub ntawv ntsig txog C. deserticola qia transcriptome tau rub tawm. 13 cov kab mob cog qoob loo 'cov ntaub ntawv noob hauv KEGG tau siv rau cov lus piav qhia siv BBH (bi-directional zoo tshaj plaws ntaus) txoj kev.

NATURAL CISTANCHE TUBULOSA CISTANCHE PHGS75% ECH 30% ACT 12%

RT-qPCR tsom xam

Tom qab kev zom nrog DNase I, kwv yees li 5ug ntawm tag nrho RNA tau hloov mus rau hauv thawj-strand cDNA ntawm qhov rov qab-txhim kho cov tshuaj tiv thaiv nrog oligo(dT)15 primers thiab GoScript Reverse Transcription System (Promega). Cov khoom cDNA ces tau diluted 10-fold nrog nuclease-dawb deionized dej ua ntej siv los ua tus qauv hauv PCR lub sijhawm. Cov cDNA tshwj xeeb tau nthuav dav los ntawm GoTaq 2-Tib RT-qPCR system (Promega) hauv ntim ntawm 20 ul. PCR amplification tau ua nyob rau ntawm qhov ntsuas kub ntawm 60 degree nrog 7500 Real-Time PCR Detection System (Applied Biosystems) raws li cov chaw tsim khoom cov lus qhia. Cov txheeb ze cov ntaub ntawv ntau ntxiv tau suav los ntawm kev sib piv lub voj voog txoj kev pib nrog cov noob "comp10579_c0" raws li tus qauv sab hauv, siv 7500 Tus Thawj Tswj software.

Primer khub rau RT-PCR tau tsim raws li hauv online software (http://primer3.ut.ee/) thiab tau teev nyob rau hauv S1 Dataset.

Cov txiaj ntsig

RNA sequencing thiab de novo transcriptome los ua ke ntawm C. deserticola fleshy qia

Ce deserticola tau siv dav los ua ib qho tseem ceeb tonic hauv Suav teb thiab Nyiv tau ntau xyoo. Txhawm rau kom tau txais kev pom thoob ntiaj teb ntawm cov noob qhia hauv C. deserticola fleshy qia, peb tau sau C. deserticola qia qauv ntawm tib tsob nroj hauv xyoo 2013 thiab 2014, raws li. Tag nrho RNAs tau muab rho tawm thiab polyA + RNAs tau ua kom huv si rau kev tsim cov tsev qiv ntawv sib txuas-kawg RNA-seq. 79,433,734 thiab 86,019,176 khub-kawg nyeem sib raug rau ze li 8 billion thiab 8.6 billion lub hauv paus ntawm qhov sib lawv liag tau siv Illumina HiSeq 2000 sequencing

platform hauv 2013- xyoo thiab 2014- xyoo qauv (Table 1). Tom qab tshem tawm cov kev sib txuas sib txuas thiab lim cov ntawv nyeem tsis zoo (saib cov ntsiab lus hauv Cov Txheej Txheem), 64,831,040 qhov zoo tshaj plaws khub-kawg nyeem hauv 2013- xyoo qauv tau siv rau de novo transcriptome los ua ke. Siv cov Trinity ib ntus sib sau ua ke [30], 51,719 noob thiab 95,787 cov ntawv sau ua ke tau tsim nrog cov ntawv sau ntev li ntawm 200 bp txog 15,698 bp. Qhov nruab nrab ntev ntawm cov ntawv sau ua ke yog 950 hauv paus thiab N50 ntev yog 1,519 hauv paus. Tus naj npawb ntawm cov ntawv sau nyob rau hauv qhov ntev sib txawv tau qhia tias 57.32% ntawm cov ntawv sau ua ke yog kwv yees li 500 bp lossis ntev dua (Daim duab 1A). High-zoo khub-kawg nyeem nyob rau hauv 2014- xyoo qauv tau mapped rau cov ntaub ntawv sau ua ke. Tsis tas li ntawd, peb pom tias tus lej sau tseg rau txhua qhov sib sau ua ke sib txawv thiab 69% ntawm cov noob nrog ib qho isoform qhia thaum 31% ntawm cov noob tau hais ob lossis ntau dua cov ntawv sau tseg (Fig 1B).

Kev nthuav qhia kom muaj nuj nqis thiab kev ua haujlwm ntawm cov ntawv sau ua ke

Gene lossis transcript abundance tau suav nrog siv pob RSEM, nyob rau hauv uas cov ntawv nyeem txuas ntxiv tau rov ua raws li cov noob sib sau ua ke lossis cov ntawv sau ua ntu zus siv Bowtie, thiab cov ntawv nyeem tau siv los ua kom muaj nuj nqis. FPKM tus nqi rau txhua tus noob lossis cov ntawv sau tau raug suav, thiab thaum kawg, peb tau txheeb xyuas 63,957 thiab 52,857 nquag qhia cov ntawv sau tseg (FPKM tus nqi Ntau dua lossis sib npaug rau 0.5) hauv C. deserticola fleshy qia qauv hauv 2{{17} }13 thiab 2014, feem. 44,776 transcripts (70.01% nyob rau hauv 2013- xyoo qauv, 84.71% nyob rau hauv 2014- xyoo qauv) feem ntau qhia nyob rau hauv ob replicates, thiab cov correlation (Pearson correlation coefficient: 0.91979) ntawm lawv cov ntaub ntawv qhia yog qhia hauv S1 Fig. Cov ntaub ntawv raw cov ntaub ntawv tau muab xa mus rau NCBI SRA database (accession naj npawb: SRX857402 thiab SRX858938). Peb tau siv cov noob caj noob ces pom nyob rau hauv 2013- xyoo qauv rau kev tshuaj xyuas ntxiv. Cov lus piav qhia ua haujlwm rau txhua daim ntawv teev lus tau hais tau siv ob txoj hauv kev. Ua ntej, tag nrho cov ntaub ntawv qhia tau raug ua raws li cov nucleotide paub (GenBank nt) thiab peptide sequence databases (GenBank nr thiab Arabidopsis peptide) cais los ntawm BLAST algorithm. Tawm ntawm 63,957 cov ntaub ntawv sau tseg,

29,220 (45.7%) tau sau tseg thiab qhia txog homology rau cov kab ke hauv ib qho ntawm peb qhov kev kawm databases nrog E-tus nqi txiav tawm 1e-20. Lub caij no, cov neeg sib tw coding cheeb tsam rau tag nrho cov ntawv sau ua ke tau kwv yees siv TransDecoder software, thiab ORFs ntev tshaj plaws rau txhua daim ntawv teev lus tau siv rau Pfam domain search. Raws li qhov tshwm sim, 21,358 (33.4%) cov ntawv sau tseg tau sau tseg raws li Pfam database. Zuag qhia tag nrho, 30,098 (47.1%) cov ntaub ntawv sau tseg tau zoo sib xws rau cov noob paub hauv cov ntaub ntawv pej xeem los ntawm kev sib txuas ob txoj hauv kev saum toj no. Daim ntawv teev cov ntawv sau ua tiav nrog cov lus piav qhia ua haujlwm tau pom hauv cov ntaub ntawv ntxiv (S2 Dataset).

Peb tau tshawb fawb saum 20 cov ntawv tshaj tawm uas tau tshaj tawm (Table 2) sib raug rau 18.99% ntawm tag nrho cov ntawv nyeem, thiab pom tias feem ntau ntawm lawv yog cov noob teb rau abiotic.

kev nyuaj siab. Dehydrin (DHNs), ib chav kawm ntawm hydrophilic thiab thermostable stress proteins uas muaj ntau cov amino acids them uas muaj nyob rau hauv pab pawg II Late Embryogenesis Abundant (LEA) tsev neeg, yog cov feem ntau qhia cov noob. Peb qhov sib txawv Dehyrin transcripts (comp28713_c0_seq1/2/4) tau kuaj pom tias muaj kev qhia ntau heev hauv cov stems uas tej zaum yuav muaj feem cuam tshuam rau kev tiv thaiv cov hlwb los ntawm kev puas tsuaj los ntawm kev ntxhov siab. Lwm yam kev ntxhov siab ntsig txog cov noob caj noob ces xws li cov protein ua rau kub nyhiab, cov kab mob ntsig txog cov protein, thiab metallothionein kuj pom tau hais tias muaj kev cuam tshuam loj heev, uas yuav cuam tshuam rau nws qhov chaw muaj sia nyob. Tsis tas li ntawd, qee cov noob caj noob ces suav nrog 26S ribosomal RNA gene (comp22329_c2_seq1), auxin-repressed / dormancy-associated protein (comp20999_c0_seq1), ADP-ribosylation factor (comp20499_ c0_seq1) kuj tau sau ntau heev.

NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev tiv thaiv kab mob PHGS75% ECH 30% ACT 12%