基于转录组测序技术的赖草根茎发育调控基因分析及应用研究

发布时间：2018-08-09 13:00

【摘要】：赖草是多年生禾本科根茎(根状茎)型植物,是异形四倍体物种(2n=4x=28)。分蘖是单子叶植物一种独特的分枝现象,在发育生物学上具有特殊的意义。通过根茎逐级分蘖即克隆生长进行无性繁殖的根茎类禾草是草原牧草种群及群落结构的重要组成部分。很多优良牧草都具有地下根茎,通过不断分蘖进而形成纵横交错的地下根茎网及与之对应的地上子代分株。赖草根茎顶芽可呈现水平和垂直两种生长方向性,本研究主要运用RNA-seq技术探讨了根茎顶芽不同生长状态下发育调控关联基因及可能的调控通路。结合形态解剖学分析和关键基因验证检测分析,试图初步揭示赖草根茎顶芽发育基因调控机理及内在分子机制。主要研究结果如下：(1)赖草水平根茎顶芽和垂直根茎顶芽转录组测序总共测得了49.01Gb的数据,每个样品Clean Data值都在7.84Gb以上,Q30碱基百分比均≥94.76%。组装后共获得了76098条Unigene。在各个数据库共注释到44769条Unigene。对Unigene库的基因结构分析,发现SSR标记5397个。KEGG注释结果提示垂直生长根茎顶芽在生长方向由水平延伸转为逆地生长后(垂直),尽管还未伸出地面,但光合相关功能基因已经提早启动表达。(2)结果表明赖草水平根茎尖和垂直根茎尖在基因表达水平上总共有4906个差异基因,其中1970个基因在水平根茎中是上调,2936个基因在垂直根茎中是上调。经过KEGG注释发现这些差异基因在以下几个通路中差异表达显著:光合作用;光合作用天线蛋白;亮氨酸代谢;谷胱甘肽代谢;生物素代谢;谷胱甘肽生物合成过程。在赖草水平根茎和垂直根茎4906个差异基因里,通过基因注释,发现其中含有32个转录因子,132个激酶。其中,有很多转录因子和激酶都与根茎的生长发育和植物抗逆性相关,这一预测结果提示:赖草水平根茎顶芽在株系扩展、延伸生长过程中,可能首先感知到生物或非生物胁,并启动相应应激代谢过程;同时,这一结果也反映出水平延伸根茎顶芽与逆地生长的垂直根茎顶芽相比,应激反馈更复杂。(3)为验证RNA-seq的结果,我们利用实时荧光定量PCR技术对其进行了检测,我们随机挑选了12个差异基因进行qRT-PCR分析,测得的结果跟RNA-Seq测序结果高度一致,说明测序所得的数据准确性很高,信息学分析的结果可信。(4)通过对赖草根茎尖进行树脂切片发现:赖草水平根茎跟垂直根茎横切片相比,水平根茎表皮层细胞较大,细胞壁较厚,组成薄壁组织环的薄壁细胞层数较多,维管束环数较多。这可能跟它们即将行使的生理功能有关,水平根茎在沙土中生长,需要穿过层层阻碍向前方生长,所以水平根茎尖有芽被保护。目前,利用生物信息学技术如RNA-seq对赖草属的根茎生长状况的研究几乎还处于零阶段。研究赖草转录组学为全面了解赖草生长发育、生理功能和抗逆相关机制提供了基础。本课题通过Illumina Hiseq2500对赖草水平生长根茎和垂直生长根茎进行转录组测序,给挖掘赖草根茎生长发育相关基因、相关代谢通路和抗逆相关基因等奠定了基础。
[Abstract]:Rhizomes of perennial grasses (rhizomes) and heteromorphic species (2n=4x=28). The tillering is a unique branching phenomenon of the mono cotyledon, and has special significance in developmental biology. The Rhizomatous Grasses of the rhizomatous growth of clonal growth through the rhizomatous tillering and clone growth are the grassland herbage population and community structure. An important part. Many excellent herbage have underground rhizomes, through continuous tillering to form a crisscross subterranean rhizome net and corresponding subsurface subplant. The top bud of Leymus rhizome can present two kinds of growth direction. This study mainly uses RNA-seq technique to explore the different growth conditions of the root bud. The regulation mechanism and internal molecular mechanism of the apical bud development of Leymus Leymus were preliminarily revealed by morphological and anatomical analysis and key gene validation detection analysis. The main results were as follows: (1) a total of 49.01 of the root buds of rhizomes and vertical root buds of Leymus Leymus were measured in total. Gb data, each sample Clean Data value is more than 7.84Gb, Q30 base percentage is more than 94.76%. assembly, a total of 76098 Unigene. in each database commented to 44769 Unigene. to the Unigene library gene structure analysis, found SSR marker 5397.KEGG annotation results show that the vertical growth root bud in the direction of growth from the level The extension turned back to the back growth (vertical), although the ground was not extended yet, the photosynthetic function gene had been started early. (2) the results showed that there were 4906 different genes in the level of rhizome and vertical rhizome of Leymus Leymus at the level of gene expression, of which 1970 genes were up-regulated in the horizontal rhizomes and 2936 genes were in the vertical rhizomes. It was up to up. After KEGG annotation, it was found that these differentially expressed genes were significantly expressed in the following pathways: photosynthesis; photosynthetic antenna protein; leucine metabolism; glutathione metabolism; biotin metabolism; glutathione biosynthesis. In the 4906 differential genes in the rhizomes and vertical rhizomes of Leymus Leymus, the findings were found by gene annotation. There are 32 transcription factors and 132 kinases. Among them, many of the transcription factors and kinases are related to the growth and stress resistance of the rhizomes. This prediction suggests that the apical bud of the rhizome of Leymus Leymus is extended in the plant line, and may first perceive raw or abiotic stresses and initiate corresponding stress metabolic processes. The results also showed that the stress feedback was more complex compared to the vertical rhizome buds of the horizontal extension rhizome and the reverse ground. (3) to test the results of RNA-seq, we used real-time fluorescence quantitative PCR to detect it. We randomly selected 12 differential causes for qRT-PCR analysis, and the results were sequenced with RNA-Seq sequencing. The results of the results were very high, indicating that the data obtained by sequencing were very accurate and the results of informatics analysis were credible. (4) through the resin section of the stem apex of the root, it was found that the horizontal rhizomes of the Leymus Leymus were larger, the cell wall was thicker than the vertical rhizome section, and the number of parenchyma cells in the parenchyma ring was more, and the vascular bundle was in the vascular bundle. There are more rings. This may be related to the physiological functions they are about to exercise. The horizontal rhizomes grow in the sandy soil and need to pass through the layers of hindrance to the front. So the tip of the horizontal rhizome is protected. At present, the study of the root growth state of the rhizome of the genus lyris by bioinformatics, such as RNA-seq, is almost zero. The transcript provides a basis for a comprehensive understanding of the growth, physiological function and anti inverse mechanism of Leymus Leymus. Through Illumina Hiseq2500, the transcriptional sequence of the rhizomes and vertical growth rhizomes of Leymus Leymus was carried out, which laid the foundation for the mining of the related genes, related metabolic pathways and resistance related genes in the rhizome of Leymus.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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