本文選題：唐古特白刺果實(shí)　切入點(diǎn)：轉(zhuǎn)錄組和表達(dá)譜測序　出處：《內(nèi)蒙古大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：唐古特白刺(Nitraria tangutorum Bobr.)隸屬于蒺藜科(Zygophyllceae)白刺屬(Nitraria L.),是一種典型的荒漠植物,具有顯著的抗干旱、鹽堿和高溫的特性,在維持荒漠生態(tài)系統(tǒng)平衡方面具有舉足輕重的作用。除了重要的生態(tài)價值,其果實(shí)作為荒漠、干旱地區(qū)少有的漿果狀核果,還具有巨大的潛在經(jīng)濟(jì)價值。但是由于缺乏遺傳信息,目前針對唐古特白刺的研究仍主要集中在形態(tài)分類、生理生態(tài)學(xué)特性和化學(xué)成分的分離及鑒定等方面。尤其是對其果實(shí)發(fā)育和成熟過程中的調(diào)控機(jī)制以及營養(yǎng)物質(zhì)的合成的分子機(jī)理的研究還很欠缺。本研究以唐古特白刺果實(shí)為實(shí)驗(yàn)材料,利用RNA-Seq技術(shù)對其進(jìn)行轉(zhuǎn)錄組測序,搭建唐古特白刺果實(shí)的遺傳信息平臺。通過對不同發(fā)育時期的果實(shí)進(jìn)行表達(dá)譜數(shù)據(jù)分析,獲得大量與果實(shí)發(fā)育和營養(yǎng)物質(zhì)合成相關(guān)的差異表達(dá)基因,為將來研究唐古特白刺果實(shí)發(fā)育過程和物質(zhì)代謝的分子機(jī)理奠定了基礎(chǔ)。主要研究結(jié)果如下：1.本研究首次運(yùn)用RNA-Seq技術(shù)對唐古特白刺果實(shí)進(jìn)行轉(zhuǎn)錄組測序,通過de novo組裝最終獲得69,306條Unigene,平均長度587 bp,搭建了該植物果實(shí)的遺傳信息平臺。對這些基因的功能進(jìn)行注釋,分別有42,929、26,809、33,363、15,537和24,592個Unigenes注釋到NCBI Nr、Swiss-Prot、GO、COG和KEGG數(shù)據(jù)庫。2.在轉(zhuǎn)錄組測序的基礎(chǔ)上對唐古特白刺果實(shí)的發(fā)育過程進(jìn)行數(shù)字基因表達(dá)譜分析,發(fā)現(xiàn)S1 vs. S2有8,240個差異表達(dá)基因(DEGs);S2 vs. S3有4,994個DEGs; S1 vs. S3有5,985個DEGs。對S1 vs. S2、S2 vs. S3和S1 vs. S3進(jìn)行GO功能富集分析發(fā)現(xiàn),分別有50,171、28,071和32,891個DEGs富集到59、58和58個GO term里。KEGG代謝通路富集分析發(fā)現(xiàn),分別有6、8和34個顯著富集的Pathway出現(xiàn)在S1 vs. S2、S2 vs. S3和SI vs. S3比較中。3.篩選出28個與植物激素相關(guān)DEGs,進(jìn)行表達(dá)模式聚類分析,將這些基因分成了4組。其中最大的一組為Group 2,包含21個DEGs,表現(xiàn)為從S1到S2下調(diào)表達(dá),S2到S3上調(diào)表達(dá)。其余3組僅含有7個DEGs,表明在唐古特白刺果實(shí)發(fā)育過程中,大多數(shù)植物激素相關(guān)的DEGs表達(dá)在發(fā)育初期和末期較高,而在中期較低。4.篩選出61個與轉(zhuǎn)錄因子相關(guān)的DEGs,進(jìn)行基因表達(dá)模式聚類分析,將這些基因同樣分成了4組。其中最大的一組為Group 1,包含42個DEGs,表現(xiàn)為從果實(shí)發(fā)育的S1到S2時期基因下調(diào)表達(dá),從S2到S3時期上調(diào)表達(dá)。另外,Group 3包含9個DEGs, Group4有6個DEGs,而Group 2僅有4個DEGs,所含的差異表達(dá)基因均較少�？梢姶蠖鄶�(shù)轉(zhuǎn)錄因子都聚類到Group 1,其表達(dá)模式與植物激素類似,即在果實(shí)發(fā)育的初期和末期基因的表達(dá)較高,而在中期較低。5.篩選出16個與植物次生代謝產(chǎn)物黃酮合成相關(guān)的DEGs和7個RPKM≥100的高表達(dá)黃酮合成途徑基因。對這些基因的表達(dá)模式進(jìn)行聚類分析,發(fā)現(xiàn)黃酮合成途徑上游的3個基因PAL、C4H和4CL均表現(xiàn)為隨著果實(shí)的發(fā)育進(jìn)程表達(dá)水平逐漸降低,其在果實(shí)發(fā)育初期的高表達(dá)可能為果實(shí)成熟時黃酮類物質(zhì)的合成積累了底物。該途徑中游的3個基因CHS, CHI和F3H的表達(dá)模式差異較大,可能導(dǎo)致形成不同種類的黃酮類化合物。黃酮合成路徑下游僅發(fā)現(xiàn)1個差異表達(dá)基因F3’H(CL7210.Contigl),該基因編碼的酶催化形成矢車菊素,這可能是唐古特白刺果實(shí)中矢車菊素含量高的原因。6.克隆了一個含有1,407 bp ORF的唐古特白刺花青素合成基因NtUFGT。利用在線分析工具及生物信息學(xué)軟件對該基因的二級結(jié)構(gòu)、亞細(xì)胞定位、信號肽、蛋白跨膜結(jié)構(gòu)域和親疏水性等特性進(jìn)行了預(yù)測。構(gòu)建了該基因的植物表達(dá)載體,并成功轉(zhuǎn)入擬南芥中,篩選獲得了轉(zhuǎn)基因擬南芥T3代純合植株。通過對轉(zhuǎn)基因植株進(jìn)行UV-B脅迫處理發(fā)現(xiàn),隨著脅迫時間的增長,轉(zhuǎn)基因擬南芥中花青素的含量高于野生型擬南芥,并呈現(xiàn)增長的趨勢。而原花青素的含量則隨著處理時間的增長,呈現(xiàn)降低的趨勢,并低于野生型擬南芥。7.測定了唐古特白刺果實(shí)不同發(fā)育時期的淀粉、蔗糖、葡萄糖和果糖的含量,以及蔗糖代謝相關(guān)酶SS、SPS、AI和NI的活性,分析了唐古特白刺果實(shí)不同發(fā)育時期這幾種糖的含量變化以及酶活性的變化情況。同時根據(jù)表達(dá)譜數(shù)據(jù)篩選了25個與蔗糖代謝相關(guān)的DEGs,13個與淀粉合成相關(guān)的DEGs,對這些基因的表達(dá)模式進(jìn)行聚類分析,探討了這些DEGs的表達(dá)變化與相關(guān)酶活性的之間的關(guān)系。
[Abstract]:N.tangutorum (Nitraria tangutorum Bobr.) belonging to the Zygophyllaceae (Zygophyllceae) Nitraria (Nitraria L.), is a kind of typical desert plants, drought resistant characteristics significantly, salinity and temperature, has a pivotal role in maintaining the balance of desert ecosystem. In addition to important ecological value. The fruit, as the desert, arid area of rare berrylike drupe, has huge potential economic value. But because of the lack of genetic information, the present research on nitrariatangutorum still focused on the morphological classification, separation and identification of physiological ecology characteristics and chemical composition especially for the fruit. The development and regulation of maturation and molecular mechanism of the synthesis of nutrients is still lacking. In this study, n.tangutorum fruit as the experimental material, the transcriptome measured by RNA-Seq Technology In order to build information platform, genetic n.tangutorum fruit. The spectrum of expression data analysis based on different developmental stages of the fruit, and get a lot of fruit growth and nutrients associated with the synthesis of differentially expressed genes, which laid the foundation for the future study of Tang Gu Nitraria fruit development process and substance metabolism of the molecular mechanism. The results are as follows: 1. the research on the application of RNA-Seq technology in n.tangutorum fruit transcriptome sequencing by de novo for the first time, the assembly eventually won the 69306 Unigene, the average length of 587 BP, built the genetic information platform of the plant fruit. The function of these genes were annotated, respectively 42929,26809,33363,15537 and 24592 Unigenes notes to NCBI Nr, Swiss-Prot, GO, COG and KEGG.2. database development process of tangutorum fruit based on transcriptome sequencing on digital gene expression S1 vs. S2 spectrum analysis, found 8240 differentially expressed genes (DEGs); S2 vs. S3 4994 DEGs; S1 vs. S3 5985 DEGs. to S1 vs. S2, S2 vs. and S3 S1 vs. S3 GO enrichment analysis showed that there were 50171,28071 and 32891 DEGs to the enrichment of 59,58 and 58 GO term.KEGG metabolic pathway enrichment analysis showed that there were 6,8 and 34 were significantly enriched Pathway appeared in S1 vs. S2, S2 vs. and S3 SI vs. S3 comparison.3. screened 28 related to plant hormone DEGs, pattern clustering analysis expression of these genes will be divided into 4 groups. One of the biggest Group group was 2, including 21 DEGs, is from S1 to S2 to S2 down expression and increased expression of S3. The remaining 3 groups containing only 7 DEGs, that in n.tangutorum fruit development process, the expression of most plant hormones related to DEGs in the development of early and late high, while in the period low.4. screen Select DEGs 61 and related transcription factors, pattern clustering analysis of gene expression, these genes are also divided into 4 groups. One of the biggest group of Group 1, including 42 DEGs, gene expression was down regulated from S1 to S2 during the fruit development of expression, from S2 to S3. In addition to rise period 3, Group contains 9 DEGs Group4, 6 DEGs and 2 Group, only 4 DEGs genes were differentially expressed with less visible. Most transcription factors are clustered into 1 Group, and its expression pattern is similar to that of plant hormones, higher expression in the early and late genes during fruit development, and in the mid lower screening.5. gene high expression of flavonoids synthesis pathway related to DEGs 16 and plant secondary metabolites synthesis of flavonoids and 7 RPKM = 100. The expression patterns of these genes by cluster analysis, 3 genes were found upstream of PAL flavonoids synthesis pathway, C4H and 4CL As the development process of fruit expression level decreased gradually and its high expression in early stage of fruit development may accumulate the substrate for the synthesis of mature fruit flavonoids. The 3 gene CHS in the middle way, differences in expression patterns of CHI and F3H may lead to larger flavonoids. Flavonoids form different kinds of downstream the synthesis path only found 1 differentially expressed genes of F3 H (CL7210.Contigl), the gene encoding the enzyme catalyzed the formation of cyanidin, this may be the n.tangutorum fruit with high content of cyanidin.6. was cloned with a 1407 BP ORF of nitrariatangutorum anthocyanin synthesis gene NtUFGT. using online analysis tools and bioinformatics software on the gene level two structure, subcellular localization, signal peptide, transmembrane domain and hydrophobic properties were predicted. The constructed surface plant gene As the carrier, and successfully transformed into Arabidopsis, transgenic Arabidopsis homozygous T3 plants. The transgenic plants were screened by UV-B stress, with the stress time increasing, the content of anthocyanin in transgenic Arabidopsis than wild type Arabidopsis, and a growing trend. But the content of procyanidins with processing time growth that showed a decreasing trend, and n.tangutorum fruit at different developmental stages of starch was determined lower than the wild type Arabidopsis.7. content of sucrose, glucose and fructose, and sucrose metabolizing enzymes SS, SPS, AI and NI activity of Nitraria fruit in different development period the content of these kinds of changes sugar and changes in enzyme activity. At the same time according to the expression of DEGs 25 and the screening of metabolism related to sucrose spectrum data, 13 DEGs associated with starch synthesis, on the expression of these genes. The relationship between the expression changes of these DEGs and the activity of related enzymes was investigated by cluster analysis.

【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張慧文;張玉;馬超美;;原花青素的研究進(jìn)展[J];食品科學(xué);2015年05期

2 張玲;吉爽爽;王蘇;王向紅;;白刺果實(shí)水溶性多糖的單糖組成研究[J];食品工業(yè);2013年11期

3 任國慧;陶然;王晨;孫欣;房經(jīng)貴;;葡萄漿果著色與UFGT和MYBA基因表達(dá)量的關(guān)系研究[J];南京農(nóng)業(yè)大學(xué)學(xué)報;2013年04期

4 劉紅亮;鄭麗明;劉青青;權(quán)富生;張涌;;非模式生物轉(zhuǎn)錄組研究[J];遺傳;2013年08期

5 郝媛媛;岳利軍;康建軍;王鎖民;;“沙漠人參”肉蓯蓉和鎖陽研究進(jìn)展[J];草業(yè)學(xué)報;2012年02期

6 岳桂東;高強(qiáng);羅龍海;王軍一;許姣卉;尹燁;;高通量測序技術(shù)在動植物研究領(lǐng)域中的應(yīng)用[J];中國科學(xué):生命科學(xué);2012年02期

7 付海輝;辛培堯;許玉蘭;劉巖;韋援教;董嬌;曹有龍;周軍;;幾種經(jīng)濟(jì)植物UFGT基因的生物信息學(xué)分析[J];基因組學(xué)與應(yīng)用生物學(xué);2011年01期

8 閆梅玲;王振平;范永;周明;孫盼;單守明;代紅軍;;蔗糖代謝相關(guān)酶在赤霞珠葡萄果實(shí)糖積累中的作用[J];果樹學(xué)報;2010年05期

9 李瀅;孫超;羅紅梅;李西文;牛云云;陳士林;;基于高通量測序454 GS FLX的丹參轉(zhuǎn)錄組學(xué)研究[J];藥學(xué)學(xué)報;2010年04期

10 姜衛(wèi)兵;徐莉莉;翁忙玲;韓健;;環(huán)境因子及外源化學(xué)物質(zhì)對植物花色素苷的影響[J];生態(tài)環(huán)境學(xué)報;2009年04期

相關(guān)博士學(xué)位論文 前3條

1 高U，

本文編號：1631963