唐古特白刺果实发育的差异表达基因分析及NtUFGT基因的克隆和功能研究

发布时间：2018-03-19 00:14

本文选题：唐古特白刺果实　切入点：转录组和表达谱测序　出处：《内蒙古大学》2016年博士论文　论文类型：学位论文

【摘要】：唐古特白刺(Nitraria tangutorum Bobr.)隶属于蒺藜科(Zygophyllceae)白刺属(Nitraria L.),是一种典型的荒漠植物,具有显著的抗干旱、盐碱和高温的特性,在维持荒漠生态系统平衡方面具有举足轻重的作用。除了重要的生态价值,其果实作为荒漠、干旱地区少有的浆果状核果,还具有巨大的潜在经济价值。但是由于缺乏遗传信息,目前针对唐古特白刺的研究仍主要集中在形态分类、生理生态学特性和化学成分的分离及鉴定等方面。尤其是对其果实发育和成熟过程中的调控机制以及营养物质的合成的分子机理的研究还很欠缺。本研究以唐古特白刺果实为实验材料,利用RNA-Seq技术对其进行转录组测序,搭建唐古特白刺果实的遗传信息平台。通过对不同发育时期的果实进行表达谱数据分析,获得大量与果实发育和营养物质合成相关的差异表达基因,为将来研究唐古特白刺果实发育过程和物质代谢的分子机理奠定了基础。主要研究结果如下：1.本研究首次运用RNA-Seq技术对唐古特白刺果实进行转录组测序,通过de novo组装最终获得69,306条Unigene,平均长度587 bp,搭建了该植物果实的遗传信息平台。对这些基因的功能进行注释,分别有42,929、26,809、33,363、15,537和24,592个Unigenes注释到NCBI Nr、Swiss-Prot、GO、COG和KEGG数据库。2.在转录组测序的基础上对唐古特白刺果实的发育过程进行数字基因表达谱分析,发现S1 vs. S2有8,240个差异表达基因(DEGs);S2 vs. S3有4,994个DEGs; S1 vs. S3有5,985个DEGs。对S1 vs. S2、S2 vs. S3和S1 vs. S3进行GO功能富集分析发现,分别有50,171、28,071和32,891个DEGs富集到59、58和58个GO term里。KEGG代谢通路富集分析发现,分别有6、8和34个显著富集的Pathway出现在S1 vs. S2、S2 vs. S3和SI vs. S3比较中。3.筛选出28个与植物激素相关DEGs,进行表达模式聚类分析,将这些基因分成了4组。其中最大的一组为Group 2,包含21个DEGs,表现为从S1到S2下调表达,S2到S3上调表达。其余3组仅含有7个DEGs,表明在唐古特白刺果实发育过程中,大多数植物激素相关的DEGs表达在发育初期和末期较高,而在中期较低。4.筛选出61个与转录因子相关的DEGs,进行基因表达模式聚类分析,将这些基因同样分成了4组。其中最大的一组为Group 1,包含42个DEGs,表现为从果实发育的S1到S2时期基因下调表达,从S2到S3时期上调表达。另外,Group 3包含9个DEGs, Group4有6个DEGs,而Group 2仅有4个DEGs,所含的差异表达基因均较少。可见大多数转录因子都聚类到Group 1,其表达模式与植物激素类似,即在果实发育的初期和末期基因的表达较高,而在中期较低。5.筛选出16个与植物次生代谢产物黄酮合成相关的DEGs和7个RPKM≥100的高表达黄酮合成途径基因。对这些基因的表达模式进行聚类分析,发现黄酮合成途径上游的3个基因PAL、C4H和4CL均表现为随着果实的发育进程表达水平逐渐降低,其在果实发育初期的高表达可能为果实成熟时黄酮类物质的合成积累了底物。该途径中游的3个基因CHS, CHI和F3H的表达模式差异较大,可能导致形成不同种类的黄酮类化合物。黄酮合成路径下游仅发现1个差异表达基因F3’H(CL7210.Contigl),该基因编码的酶催化形成矢车菊素,这可能是唐古特白刺果实中矢车菊素含量高的原因。6.克隆了一个含有1,407 bp ORF的唐古特白刺花青素合成基因NtUFGT。利用在线分析工具及生物信息学软件对该基因的二级结构、亚细胞定位、信号肽、蛋白跨膜结构域和亲疏水性等特性进行了预测。构建了该基因的植物表达载体,并成功转入拟南芥中,筛选获得了转基因拟南芥T3代纯合植株。通过对转基因植株进行UV-B胁迫处理发现,随着胁迫时间的增长,转基因拟南芥中花青素的含量高于野生型拟南芥,并呈现增长的趋势。而原花青素的含量则随着处理时间的增长,呈现降低的趋势,并低于野生型拟南芥。7.测定了唐古特白刺果实不同发育时期的淀粉、蔗糖、葡萄糖和果糖的含量,以及蔗糖代谢相关酶SS、SPS、AI和NI的活性,分析了唐古特白刺果实不同发育时期这几种糖的含量变化以及酶活性的变化情况。同时根据表达谱数据筛选了25个与蔗糖代谢相关的DEGs,13个与淀粉合成相关的DEGs,对这些基因的表达模式进行聚类分析,探讨了这些DEGs的表达变化与相关酶活性的之间的关系。
[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.

【学位授予单位】：内蒙古大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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