利用RNA-seq解析甘蓝型油菜根尖致死突变的分子机理

发布时间：2018-06-23 16:35

本文选题：甘蓝型油菜(Brassica + napus)　；参考：《华中农业大学》2017年硕士论文

【摘要】：甘蓝型油菜(Brassica napus L.)是油菜三大类型之一,由甘蓝(B.olerac ea CC,2n=18)与白菜(B.rapa,AA,2n=20)在大约7500年前经过自然种间杂交再双二倍体化形成异源多倍体。远古时代油菜的细胞学、分子遗传学及育种研究都已经取得了重大的研究进展,但调控根生长发育的分子机理研究较少,有必要建立油菜根尖发育阶段的转录组信息库。本研究旨在于油菜根尖两个不同发育阶段:根尖致死阶段和根尖正常阶段,进行转录组测序,分析根尖发育不同阶段的表达谱。探究调控根生长发育的分子机理,可为选育优良品质的油菜品种提供参考信息。主要研究结果如下:1、体式显微镜下根尖不同发育阶段的形态学观察。根尖致死和根尖正常两个材料在种子萌发过程中的胚发育早期,出现表型差异:突变体材料表型明显,根尖先出现一个黑点,并逐渐变大,最终根尖停止发育。2、本研究利用Illumina Hi Seq 2000双末端二代测序技术,获得了44.5GB的转录组数据,采用Hisat2软件进行比对,结果显示Hisat的reads比对率均在90%左右。3、本研究获得了根尖致死和根尖正常两个发育阶段的表达谱数据库。八个样品共有47968个基因表达。分析表明,差异基因第一个阶段,根尖致死较根尖正常上调表达1864个基因,下调表达1447个基因;第二个阶段根尖致死较根尖正常上调表达425个基因,下调表达488个基因;两个阶段共同上调表达119个基因,共同下调表达53个基因。4、本研究对两个阶段的差异基因,进行了GO功能分类、GO通路显著性富集及KEGG pathway富集分析。两个阶段的差异基因在GO功能分类中主要分布在catalytic activity、binding、cell、organelle、cell part、metabolic process、cellular process、single-organism process、regulation of biological process、response to stimulus、localization、以及biological regulation条目上。在GO数据库中,两个阶段共同的差异基因显著性富集到两个路径,分别为nucleic acid binding transcription factor activity(GO:0001071)路径和structural constituent of cell wall路径。将两个阶段共同差异基因比对到KEGG数据库中,获得了2条显著性代谢通路,分别为Steroidbiosynthesis(KO:ko00100)类固醇合成途径,alpha-Linolenic acid metabolism(ko:00592)代谢途径。5、分析发现有四个差异基因的GO功能注释为cell killing,这四个基因分别为Bna A05g03400D、Bna C04g49050D、Bna A02g17520D、Bna C02g23400D。将其比对到拟南芥数据库中,发现Bnaco2g23400和Bna A02g17520是同源基因,Bna A05g03400和Bna C04g49050是同源基因,且这四个基因的GO功能注释与本研究中的表型性状相吻合,这四个基因被列为候选基因。6、基因(Bna A02g17520D)在拟拟南芥中(ATG37870)为磷酸烯醇丙酮酸羧基酶,该酶是糖异生的关键酶,受MC9依赖性蛋白活性的影响是MC9的底物,具有与细胞死亡相关降解的功能。本研究中GO富集结果差异基因富集到内质网蛋白加工(Protein processing in endoplasmic reticulum Ko0441)诱导内质网相关性降解这条代谢途径上,两者相互验证。因此,推测该基因在油菜中可能具有导致细胞致死的功能。
[Abstract]:Brassica napus L. (Brassica napus) is one of the three major types of rapeseed. It has been studied greatly in the cytology, molecular genetics and breeding of rapeseed in the ancient times, by B.olerac EA CC (2n=18) and cabbage (B.rapa, AA, 2n=20) about 7500 years ago by interspecific hybridization and diploid formation of heterogenous polyploid. However, there are few studies on the molecular mechanism of regulating root growth and development. It is necessary to establish a transcriptional database for the development stage of rapeseed root tip. This study aims at two different developmental stages of rapeseed root apex: the root tip death stage and the normal stage of the root apex, and the transcriptional sequence is carried out to analyze the expression profiles at different stages of root apex development. The molecular mechanism of development can provide reference information for the selection of fine quality rapeseed varieties. The main results are as follows: 1, morphological observation of different developmental stages of root apex under the body microscope. In the early development of the embryo, two materials of root tip death and root apex were in the early stage of embryo development, and there were phenotypic differences: the phenotype of the mutant material was obvious, The root tip first appeared a black spot, and gradually became larger, finally the root tip stopped developing.2. This study obtained the 44.5GB transcriptional data using the Illumina Hi Seq 2000 double terminal two generation sequencing technology, and the Hisat2 software was compared. The results showed that the reads ratio of Hisat was about 90%.3. This study obtained the root tip death and the root apex normal two. A total of 47968 genes were expressed in eight samples. The analysis showed that the first stage of the difference gene, 1864 genes were up-regulated and 1447 genes were down regulated, and 1447 genes were downregulated in the root tip. The second stages of root tip death were up to 425 genes, 488 genes were downregulated, and two steps were downregulated. The 119 genes were up-regulated and 53 genes.4 were down regulated together. The GO function classification, the significant enrichment of the GO pathway and the KEGG pathway enrichment analysis were carried out on the difference genes of the two stages. The difference genes in the two stages were mainly distributed in the catalytic activity, binding, cell, organelle, cell, etc. The bolic process, the cellular process, the single-organism process, the regulation of biological process, the response to, and the entries. The Y (GO:0001071) path and the structural constituent of cell wall path. 2 significant metabolic pathways were obtained by comparing the two stages of the common differential gene into the KEGG database, respectively, the Steroidbiosynthesis (KO:ko00100) synthesis pathway, the alpha-Linolenic acid metabolic pathway, and the analysis of four The GO function annotation of the differential gene is cell killing, and these four genes are Bna A05g03400D, Bna C04g49050D, Bna A02g17520D and Bna C02g23400D.. The four genes are listed as candidate genes.6, and the gene (Bna A02g17520D) in Arabidopsis (ATG37870) is a phosphoenolpyruvate carboxylase, which is the key enzyme of sugar isogenesis. The effect of MC9 dependent protein activity is the sole of MC9 and has the function of degradation related to cell death. In this study, G The difference genes of the O enrichment results are enriched in the endoplasmic reticulum protein processing (Protein processing in endoplasmic reticulum Ko0441) induced endoplasmic reticulum related degradation, which can be verified by each other. Therefore, it is presumed that the gene may have the function of causing cell death in rape.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4

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