摘 要 草蓯蓉為東北道地藥材,具有補(bǔ)腎壯陽,潤(rùn)腸通便,止血的功效,草蓯蓉中含有草蓯蓉多糖、草蓯蓉環(huán)烯醚萜苷、草蓯蓉酸等活性成分,具有豐富的藥用價(jià)值。目前對(duì)草蓯蓉的研究多集中于有效成分,藥理性等方面,在轉(zhuǎn)錄組及功能基因方面的內(nèi)容較少。為進(jìn)一步了解大興安嶺草蓯蓉的轉(zhuǎn)錄組,豐富其遺傳信息利用高通量測(cè)序技術(shù)獲得大興安嶺草蓯蓉種子的轉(zhuǎn)錄組信息。利用轉(zhuǎn)錄組測(cè)序獲得Unigene序列,將Unigene與七大功能數(shù)據(jù)庫(kù)注釋進(jìn)行比對(duì)注釋、SSR分析、CDS預(yù)測(cè),得到草蓯蓉轉(zhuǎn)錄組的信息。結(jié)果顯示,共獲得18.87 G的CleanData,各樣本的有效數(shù)據(jù)量分布在5.4~7.06 Gb,Q30堿基分布在95.66%~97.45%,平均GC含量為49.35%。拼接出Unigene 57 799條,總長(zhǎng)度為48 308 661 bp,平均長(zhǎng)度為835.8 bp。大興安嶺草蓯蓉與油菜(Brassica napus)序列相似度最高,47 035個(gè)(81.38%)基因注釋到非冗余蛋白序列數(shù)據(jù)庫(kù)(NR), "33 653個(gè)(58.22%)基因注釋到蛋白序列數(shù)據(jù)庫(kù)(Swissprot),13 174個(gè)(22.79%)基因注釋到京都基因與基因組百科全書數(shù)據(jù)庫(kù)(KEGG),27 886個(gè)(48.25%)基因注釋到真核生物蛋白相鄰類的聚簇?cái)?shù)據(jù)庫(kù)(KOG), "42 506個(gè)(73.54%)基因注釋到直系同源蛋白分組比對(duì)數(shù)據(jù)庫(kù)(eggNOG),30 928個(gè)(53.51%)基因注釋到基因本體論數(shù)據(jù)庫(kù)(GO),29 642個(gè)(51.28%)基因注釋到蛋白家族數(shù)據(jù)庫(kù)(Pfam)。預(yù)測(cè)出SSRs 11 031個(gè),包含SSRs的Unigene有57 799條,包含大于1個(gè)SSRs的Unigene有1 886條,復(fù)合型SSRs943個(gè)。預(yù)測(cè)出 "53 033條CDS序列,其中數(shù)據(jù)庫(kù)比對(duì)方法預(yù)測(cè)出47 140條,ESTScan預(yù)測(cè)出5 893條。研究結(jié)果豐富了大興安嶺草蓯蓉的轉(zhuǎn)錄組數(shù)據(jù),為深入研究大興安嶺[JP3]草蓯蓉生物學(xué)特性及分子機(jī)制等提供參考,也有利于其資源開發(fā)和保護(hù)利用。[JP]
關(guān)鍵詞 草蓯蓉;全長(zhǎng)轉(zhuǎn)錄組測(cè)序;大興安嶺;代謝通路;生物信息分析
草蓯蓉(Bosehniakia rossica)為列當(dāng)科草蓯蓉屬植物,多分布于中國(guó)黑龍江、吉林、內(nèi)蒙古等地區(qū)。全草入藥,味甘咸,性溫,功效補(bǔ)腎壯陽,潤(rùn)腸通便,止血,用于腎虛陽萎、腰關(guān)節(jié)冷痛、便秘等,具有豐富的藥用價(jià)值[1-5]。草蓯蓉種子中含有草蓯蓉多糖、草蓯蓉環(huán)烯醚萜苷,草蓯蓉苷、草蓯蓉酸等活性成分[6-8],具有抗腫瘤、抗炎、抗氧化、保肝等作用[9-10]。
董學(xué)花等[11]研究發(fā)現(xiàn),草蓯蓉環(huán)烯醚萜苷能抑制人肝癌HepG2細(xì)胞活力,降低細(xì)胞黏附率,還能顯著下調(diào)TGF-β1誘導(dǎo)后HepG2中MMP2、MMP7、MMP9、Snail、Slug蛋白表達(dá),說明草蓯蓉環(huán)烯醚萜苷具有抗腫瘤作用;李香丹等[12]對(duì)采用四氯化碳植物油混懸液建立肝纖維化模型大鼠,與模型組相比,草蓯蓉環(huán)烯醚萜苷給藥組能明顯降低肝組織中的ALT、AST、TBIL水平,減輕肝纖維化程度,說明草蓯蓉環(huán)烯醚萜苷對(duì)肝損傷有一定的保護(hù)作用;鄭峰等[13]研究發(fā)現(xiàn),經(jīng)草蓯蓉環(huán)烯醚萜苷灌胃后可使肝癌大鼠血清中的MDA、GST水平降低,增加SOD活性,說明草蓯蓉化學(xué)醚萜苷能通過抗氧化作用進(jìn)而發(fā)揮抗腫瘤作用;此外,草蓯蓉多糖能抑制炎癥因子的表達(dá),如IL-β、IL-6、TNF-α等,還能降低LPS誘導(dǎo)的RAW264.7巨噬細(xì)胞中COX-2、MyD88蛋白含量[14]。
草蓯蓉最早記載于唐《新修本草》[15]。大興安嶺以淺山丘陵為主,屬中溫帶大陸性季風(fēng)氣候。草蓯蓉性耐寒冷,喜愛冷涼氣候,大興安嶺的主要生態(tài)因子都適宜草蓯蓉生長(zhǎng),故草蓯蓉成為大興安嶺道地藥材之一[16-18]。有關(guān)草蓯蓉分子生物學(xué)、轉(zhuǎn)錄組和功能基因的相關(guān)信息較少,在一定程度上制約了草蓯蓉研究的進(jìn)展。[JP3]因此利用高通量測(cè)序技術(shù)對(duì)草蓯蓉種子進(jìn)行分析,為擴(kuò)大草蓯蓉資源,進(jìn)一步探索其功能基因信息提供理論依據(jù)。
1 材料與方法
1.1 試驗(yàn)材料
大興安嶺草蓯蓉轉(zhuǎn)錄組測(cè)序材料于2022年9月在大興安嶺地區(qū)采集,采集多個(gè)植株生長(zhǎng)成熟的草蓯蓉種子,用錫紙包裹并快速放入液氮處理,置于-80 ℃保存?zhèn)溆谩?/p>
1.2 大興安嶺草蓯蓉種子RNA抽提和文庫(kù)構(gòu)建
用TRIzo(ThermoScientific,貨號(hào)A33251)提取總RNA,采用NanoDrop2000分光光度計(jì)(ThermoScientific, USA)檢測(cè)RNA純度和定量,并用Agilent2100生物分析儀(AgilentTechnologies, SantaClara, CA, USA)評(píng)估RNA完整性。然后根據(jù)說明書使[JP3]用VAHTS Universal V6RNA-seqLibraryPrep試劑盒(Vazyme-innovation in enzyme technology,USA)構(gòu)建轉(zhuǎn)錄組文庫(kù)。[JP]
1.3 轉(zhuǎn)錄組測(cè)序與組裝
文庫(kù)質(zhì)量經(jīng)Agilent 2100生物分析儀質(zhì)檢合格后,使用IlluminaNovaseq6000測(cè)序平臺(tái)進(jìn)行測(cè)序,生成150 bp的雙端序列。建庫(kù)測(cè)序先經(jīng)過種子總RNA提取與RNA的質(zhì)量檢測(cè),再對(duì)RNA進(jìn)行富集、片段化逆轉(zhuǎn)錄生成cDNA,最后PCR擴(kuò)增完成上級(jí)測(cè)序,此步驟及部分?jǐn)?shù)據(jù)分析由上海歐易生物醫(yī)學(xué)科技有限公司完成。高通量測(cè)序得到的原始圖像數(shù)據(jù)文件經(jīng)堿基識(shí)別(BaseCalling)分析轉(zhuǎn)化為原始測(cè)序序列,稱之為Raw Data或Raw Reads,結(jié)果以FASTQ文件格式存儲(chǔ),其中包含測(cè)序序列(reads)的序列信息以及其對(duì)應(yīng)的測(cè)序質(zhì)量信息。通過測(cè)序,得到大量的樣本雙端測(cè)序數(shù)據(jù)。采用fastp軟件對(duì)原始數(shù)據(jù)進(jìn)行質(zhì)量預(yù)處理,首先對(duì)原始數(shù)據(jù)進(jìn)行質(zhì)控并去除接頭,再?gòu)?′端及5′端以不同方式去除低質(zhì)量堿基,最終得到高質(zhì)量的cleanreads。用Trinity軟件paired-end的拼接方法得到Transcript序列,根據(jù)序列相似性以及長(zhǎng)度,挑選出最長(zhǎng)的序列作為Unigene;之后再利用CD-HIT軟件聚類去冗余得到最終的Unigene,以此作為后續(xù)分析的參考序列。
1.4 Unigene功能注釋
對(duì)NR、COG、KOG、GO、Swiss-Prot、eggNOG、KEGG數(shù)據(jù)庫(kù)注釋,采用diamond軟件進(jìn)行比對(duì),取elt;1e-5的注釋,篩選具有最高序列相似性的蛋白,從而得到功能注釋信息。對(duì)于Pfam數(shù)據(jù)庫(kù)注釋,采用HMMER軟件和蛋白家族模型比對(duì),從而篩選出得分最高的家族。
1.5 CDS預(yù)測(cè)
將Unigene序列與NR、SwissProt和KOG數(shù)據(jù)庫(kù)作對(duì)比,直到與所有蛋白數(shù)據(jù)庫(kù)對(duì)比完,剩余對(duì)比不上的Unigene的編碼區(qū)采用ESTScan(3.0.3) 軟件預(yù)測(cè),獲得其編碼區(qū)的核酸序列(序列方向5′-gt;3′)和氨基酸序列。
1.6 SSR分析
利用MISA進(jìn)行SSR預(yù)測(cè),然后利用Primer3將獲得的大興安嶺草蓯蓉種子SSR位點(diǎn)的序列以退火溫度60 ℃±2 ℃、引物長(zhǎng)度20 bp左右、PCR產(chǎn)物長(zhǎng)度100~300 bp 為條件進(jìn)行引物設(shè)計(jì)。
2 結(jié)果與分析
2.1 轉(zhuǎn)錄組測(cè)序和denovo組裝
對(duì)大興安嶺草蓯蓉種子樣本進(jìn)行轉(zhuǎn)錄組測(cè)序,每個(gè)樣本重復(fù)3次,共獲得18.87 Gb的CleanData,各樣本的有效數(shù)據(jù)量分布在5.4~7.06 Gb,Q30堿基分布在95.66%~97.45%,平均GC含量為49.35%。上述數(shù)據(jù)說明獲得的轉(zhuǎn)錄組數(shù)據(jù)質(zhì)量較好,可用于后續(xù)的分析處理(表1)。
使用Trinity將cleanreads進(jìn)行denovo組裝成轉(zhuǎn)錄本,去冗余后共獲得57 799條Unigene,總長(zhǎng)48 308 661 bp,平均長(zhǎng)度為835.8 bp,N50為 "1 075 bp;其中長(zhǎng)度大于等于500 bp的有35 255條序列,占比61.00%;15 752條序列長(zhǎng)度大于等于1 000 bp,占比27.25%,見圖1。
2.2 Unigene功能注釋
大興安嶺草蓯蓉組裝后的Unigene序列與7個(gè)常見數(shù)據(jù)庫(kù)比對(duì),見表2。其中注釋到Unigene最多的數(shù)據(jù)庫(kù)為NR數(shù)據(jù)庫(kù),有47 035條,占 "81.38%,KEGG數(shù)據(jù)庫(kù)被注釋到的Unigene最少,有13 174條,占22.79%。
為了解大興安嶺草蓯蓉種子的相近物種情況,將其轉(zhuǎn)錄組序列與NR庫(kù)數(shù)據(jù)比對(duì),匹配較多的物種主要有油菜(Brassica napus)、芝麻(Sesamum indicum)、蕓苔屬植物(Brassica rapa)、獨(dú)腳金(Striga asiatica)、紫花風(fēng)鈴木(Handroanthus impetiginosus),分別占12.09%、12.00%、8.83%、6.49%、5.84%。結(jié)果顯示大興安嶺草蓯蓉種子與油菜(Brassica napus)具有的同源序列匹配度比例最高, 見圖2。
2.3 KEGG功能注釋
KEGG數(shù)據(jù)庫(kù)注釋結(jié)果顯示有關(guān)大興安嶺草蓯蓉糖類的合成與代謝的Unigene被注釋335條,有關(guān)萜類和多酮化合物代謝的Unigene有263條,這3類Unigene可能與大興安嶺草蓯蓉的活性成分的合成與代謝有關(guān),見圖3。根據(jù)KEGG數(shù)據(jù)庫(kù)比對(duì),篩選出參與大興安嶺草蓯蓉種子活性成分合成通路的 Unigene,包括132條Unigene涉及萜類骨架生物合成,129條Unigene涉及精氨酸生物合成,128條Unigene涉及苯丙氨酸、酪氨酸和色氨酸生物合成,90條Unigene涉及類固醇生物合成,87條Unigene涉及泛醌和萜類醌生物合成,85條Unigene涉及葉酸生物合成,64條Unigene涉及賴氨酸生物合成,60條Unigene涉及三萜、哌啶和吡啶生物合成,56條Unigene涉及異喹啉生物堿生物合成,40條Unigene涉及類胡蘿卜素生物合成,38條Unigene涉及單環(huán)類生物合成,30條Unigene涉及類黃酮生物合成,13條Unigene涉及二萜類生物合成,9條Unigene涉及單萜類生物合成。
2.4 GO功能注釋
對(duì)大興安嶺草蓯蓉轉(zhuǎn)錄組Unigene進(jìn)行GO功能注釋,共分類到BP、CC和MF" 3個(gè)GO類別的44個(gè)條目中。其中,在第一大類BP中,共包含21個(gè)條目,其中Cellularprocess(細(xì)胞過程)和Metabolicprocess(代謝過程)基因數(shù)量最多;在第二大類Cellularcomponent(細(xì)胞成分)中,共包含有13個(gè)條目,其中Cell(細(xì)胞)、Cellpart(細(xì)胞組分)的基因數(shù)量最多;第三大類MF中,共包含有10個(gè)條目,Binding(結(jié)合因子)和Catalyticactivity(催化因子活性)的基因數(shù)量最多,見圖4。
2.5 KOG功能注釋
KOG數(shù)據(jù)庫(kù)注釋了60 500個(gè)轉(zhuǎn)錄本,按照KOG功能分類可分為25個(gè)組,見圖5。其中,“一般功能預(yù)測(cè)”的轉(zhuǎn)錄本最多,共4 778個(gè);“蛋白質(zhì)翻譯后修飾、蛋白周轉(zhuǎn)、分子伴侶”的轉(zhuǎn)錄本有3 380個(gè);“翻譯、核糖體結(jié)構(gòu)與生物發(fā)生”的轉(zhuǎn)錄本有2 998個(gè)。此外,發(fā)現(xiàn)“次生代謝物生物合成、轉(zhuǎn)運(yùn)與代謝”等相關(guān)的轉(zhuǎn)錄本有806個(gè),這些轉(zhuǎn)錄本可能與大興安嶺草蓯蓉中有效成分的合成途徑有關(guān)。
2.6 編碼序列(CDS)分析
對(duì)大興安嶺草蓯蓉轉(zhuǎn)錄組所有Unigene的CDS序列進(jìn)行預(yù)測(cè),共預(yù)測(cè)出53 033條CDS序列,其中數(shù)據(jù)庫(kù)比對(duì)方法預(yù)測(cè)出47 140條。對(duì)預(yù)測(cè)的CDS序列長(zhǎng)度進(jìn)行整合,其中長(zhǎng)度≥500 nt的序列占總序列數(shù)的比例為48.32%,長(zhǎng)度≥ "1 000 nt的序列占總序列數(shù)的比例為18.69%。ESTScan預(yù)測(cè)出5 893條,對(duì)預(yù)測(cè)的CDS序列長(zhǎng)度進(jìn)行整合,其中長(zhǎng)度≥500 nt的序列占總序列數(shù)的比例為16.14%,長(zhǎng)度≥1 000 nt的序列占總序列數(shù)的比例為1.49%,見圖6和圖7。
2.7 簡(jiǎn)單重復(fù)序列(SSR)分析
根據(jù)組裝結(jié)果,使用MISA軟件對(duì)Unigene的SSR進(jìn)行檢測(cè),將SSR統(tǒng)計(jì)結(jié)果按重復(fù)類型分為6類,共11 031條SSR。其中最豐富的重復(fù)類型為單堿基重復(fù),共有5 281條(占47.87%);其次為雙堿基重復(fù)和三堿基重復(fù)類型,分別為 "2 998條(占27.18%)和2 497條(22.64%);其余為四、五和六堿基重復(fù)類型,共255條(占 "2.31%),見表3。根據(jù)引物設(shè)計(jì)條件獲得了 "10 088條符合條件的引物(表4),這將為后續(xù)草蓯蓉分子標(biāo)記的開發(fā)提供參考。
3 討論與結(jié)論
轉(zhuǎn)錄組分析能獲得植物的功能基因從而了解植物在生長(zhǎng)發(fā)育階段的調(diào)節(jié)機(jī)制,草蓯蓉轉(zhuǎn)錄組測(cè)序結(jié)果顯示,大興安嶺草蓯蓉種子共獲得 "18.87" Gb CleanData,經(jīng)過建庫(kù)測(cè)序與生物信息分析得知Q30達(dá)到97.45%,GC含量為 "49.35%,表明大興安嶺草蓯蓉種子轉(zhuǎn)錄組測(cè)序數(shù)據(jù)質(zhì)量?jī)?yōu)良。通過denovo組裝,共獲得57 799條轉(zhuǎn)錄本,其中序列長(zhǎng)度大于等于500 bp的有 "35 255條,平均長(zhǎng)度為835.80 bp,N50為1 075 bp,表明大興安嶺草蓯蓉轉(zhuǎn)錄組序列組裝質(zhì)量較高。序列比對(duì)顯示大興安嶺草蓯蓉種子與油菜(Brassica napus)和芝麻(Sesamumin dicum)親緣關(guān)系最為接近。通過KEGG功能注釋,其中有關(guān)大興安嶺草蓯蓉糖類的合成與代謝的Unigene被注釋335條,有關(guān)萜類和多酮化合物代謝的Unigene有263條,有關(guān)其他次生代謝物的生物合成的Unigene有342條。草蓯蓉發(fā)揮藥效的主要化學(xué)成分為草蓯蓉多糖等,糖類作為物質(zhì)代謝的碳骨架能為植物提供能量,研究表明多糖類化合物參與了細(xì)胞各種生命現(xiàn)象的調(diào)節(jié),具有多種生理活性[19],因此對(duì)該類Unigene的挖掘和分析具有重要的研究意義。萜類化合物廣泛分布于自然界,具有保護(hù)植物細(xì)胞膜,刺激植物產(chǎn)生內(nèi)源激素并且保護(hù)植物免受強(qiáng)光的傷害等作用[20],其中三萜皂苷不僅能為植物抵御病原體還具有多種藥理活性[21],轉(zhuǎn)錄組測(cè)序結(jié)果顯示有關(guān)萜類和多酮化合物代謝的Unigene有263條,這些Unigene可能與大興安嶺草蓯蓉種子中萜類成分的合成與代謝有關(guān)。目前,SSR標(biāo)記已在蘋果等不同抗性植物的分子遺傳學(xué)檢測(cè)中得到很好的應(yīng)用,本研究共檢測(cè)到11 031條SSR,其中最豐富的重復(fù)類型為單堿基重復(fù),共有5 281條,為資源利用與藥材鑒定提供參考。大興安嶺草蓯蓉種子在調(diào)節(jié)碳代謝、氨基酸代謝、能量代謝和脂質(zhì)代謝等方面的基因可能是在生長(zhǎng)發(fā)育時(shí)期應(yīng)對(duì)低溫脅迫的基礎(chǔ)。本研究對(duì)大興安嶺草蓯蓉種子進(jìn)行了轉(zhuǎn)錄組測(cè)序分析,獲得大量轉(zhuǎn)錄水平數(shù)據(jù),豐富了對(duì)草蓯蓉道地產(chǎn)區(qū)基因的認(rèn)識(shí), 為今后草蓯蓉功能基因的進(jìn)一步探索提供了一定的數(shù)據(jù)參考, 同時(shí)也為日后基因工程技術(shù)對(duì)草蓯蓉進(jìn)行遺傳改良奠定了理論基礎(chǔ)。
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Full-length Transcriptome Sequencing and Bioinformatics Analysis of Boschniakia" rossica in Daxing’an Mountains
Abstract Boschniakia rossica is a valuable medicinal materials native to northeastern China,known for its therapeutic effect in strengthening kidney function,combating constipation, and promoting hemostasis.It contains active compounds such as Boschniakia rossica polysaccharides, Iridoid glucosides,Boschniakia rossica acid, all contributing to its significant medicinal value.To further explore the transcriptome of stanche from Daxing’an Mountains and enhance its genetic database, we conducted a full-length transcriptomic analysis of Boschniakia rossica seeds using high-throughput sequencing technology.Through transcriptomes squencing we obtained Unigene sequence,followed by functional annotation, SSR (Simple Sequence Repeat) analysis and CDS (Coding Sequence) prediction, and an in-depth analysis of genetic information.The results showed that the a total of 18.87 Gb" of clean data was obtained, with the effective data per sample ranging from 5.4 to 7.06 Gb.The Q30 base quality score ranged from 95.66%" to 97.45%, and the average GC content was 49.35%.We identified 57 799 Unigen sequnces, with a total length of 48 308 661 bp and an average length of" "835.8 bp.The Boschniakia rossica seeds transcriptome showed the highest sequence similarity to Brassica napus.Functional annotations revealed that 47 035(81.38%) genes were annotated in the NR database," "33 653(58.22%) in Swissprot, 13 174(22.79%) in KEGG, 27 886(48.25%) in KOG, 42 506 "(73.54%) in eggNOG, 30 928(53.51%) in GO, 29 642(51.28%) in Pfam.11 031 SSRs were predicted, with" "57 799 Unigene containing SSRs, 1 886 Unigene containing more than 1SSRs, and compound SSRs 943 predicted.A total of 53 033 CDS sequences were predicted, with 47 140 were predicted by the database alignment and 5 893 by ESTScan.These findings enrich the transcriptome data of Boschniakia rossica, and provide a reference for further studying the biological characteristics and molecular mechanism of Boschniakia rossica.This study also lays a foundation for the sustainable exploitation, protection, and utilization of Boschniakia rossica resources.
Key words Boschniakia rossica; Full-length transcriptome sequencing;Daxing’an Mountains;Metabolic pathway; Bioinformatics analysis