烟草CV58抗TMV基因的克隆及TMV胁迫下microRNA的表达谱分析

发布时间：2018-03-26 09:46

本文选题：烟草　切入点：烟草花叶病毒　出处：《沈阳农业大学》2017年硕士论文

【摘要】：作为烟草花叶病毒属(Tobawovirus)的代表种,烟草花叶病毒(Tobacco mosaic virus,TMV)是烟草中广泛传播的一种病毒,严重影响烟草的品质和产量。因此,筛选高抗TMV的烟草材料,鉴定抗性基因并分析其表达特征具有重要意义。另一方面,在植物抗病毒防卫反应的转录后基因沉默过程中,microRNA(miRNA)起着重要的作用,它可介导具有同源序列的外源病毒基因miRNA裂解或抑制其翻译。烟草CV58(Vicotiana tabacum cv.CV58)作为一种高抗TMV的重要种质资源,对其开展miRNA的鉴定与功能分析,有助于阐释品种的抗性信号通路。本研究主要研究成果如下:1.抗病性鉴定表明,烟草研究所自主培育烟草品种CV58在感染TMV后3天出现明显的HR,形成类似N基因的枯斑症状,限制病毒扩展,为鉴定此抗性基因,首先通过PCR同源克隆获得烟草CV58中抗性基因的CDS序列及基因组序列,序列分析显示含3435 bp核苷酸,编码1145个氨基酸,序列同源性比较显示与N基因有99%的相似性。为进一步验证CV58抗TMV特性由N基因所致,构建CRISPR/Cas9敲除载体,分别获得靶位点序列缺失2个和3个碱基的纯合突变植株,突变烟草植株在接种TMV后显示失去对TMV的抗性而出现花叶症状,且其温度敏感性一并丧失。再通过农杆菌介导的方法将该基因转入感病烟草植株K326中进行过表达,接种TMV后同样出现类似N基因的枯斑,且具有温度敏感性。综上所述,最终确定烟草CV58的抗性来源于N基因。2.CV58植株分别接种TMV或PBS,提取总RNA经Illumina Hiseq 2000测序系统检测miRNA,与己知数据库比对筛选出两样本RNA中差异表达的miRNA。将差异表达的miRNA分为上调组和下调组,分别对选定的miRNA进行靶基因预测;对交集的靶标基因集合分别进行GO富集分析和KEGG分析,预测可能的信号通路。表达谱分析显示:在差异表达的己知329个miRNA中,(上调211个,下调118个,p0.05),256个具有显著差异的 miRNA(Fold-change1.00 的有 184 个,Fold-change-1.00 的 72 个)。TMV抗性相关的miRNA差异表达谱信息学分析靶标基因指向植物-病源菌互作、细胞发育和分裂、抗性基因R介导的吞噬等7条通路,为后续的验证miRNA在植物抗病毒机制中的作用提供依据。3.以TMV侵染烟草CV58的叶片为材料,利用GeNorm和NorFinder程序对包括9个成熟miRNAs和5个传统的看家基因14个候选内参基因的稳定性进行评价。结果显示在TMV侵染胁迫下,在烟草CV58组织、叶片中,成熟miRNA定量最合适的单个内参基因分别为L25、miR159,最合适的内参基因组合为L25,Ntubc2。
[Abstract]:As a representative species of the genus Tobawovirus, tobacco mosaic virus (TMV) is a widely spread virus in tobacco, which seriously affects the quality and yield of tobacco. It is important to identify resistance genes and analyze their expression characteristics. On the other hand, microRNAs miRNAs play an important role in the post-transcriptional gene silencing of plant anti-virus defense response. It can mediate the miRNA cleavage or inhibit the translation of foreign virus gene with homologous sequence. As an important germplasm resource of high resistance to TMV, tobacco CV58(Vicotiana tabacum cv. CV58 has been identified and analyzed for its miRNA function. The main results of this study are as follows: 1. The identification of resistance to disease in tobacco varieties showed that the disease resistance of tobacco varieties CV58 was obvious in 3 days after infection with TMV, and the symptoms were similar to those of N gene. In order to identify the resistance gene, the CDS sequence and genome sequence of the resistance gene in tobacco CV58 were obtained by PCR homologous cloning. Sequence analysis showed that the resistance gene contained 3435 BP nucleotides and encoded 1145 amino acids. Sequence homology analysis showed that there was a 99% similarity with N gene. In order to further verify that the resistance of CV58 to TMV was caused by N gene, CRISPR/Cas9 knockout vector was constructed, and homozygous mutant plants with 2 and 3 bases missing target sites were obtained, respectively. After inoculation with TMV, the mutant tobacco plants showed mosaic symptoms due to loss of TMV resistance, and their temperature sensitivity was also lost. The gene was expressed in susceptible tobacco plant K326 by Agrobacterium tumefaciens-mediated method. After inoculation with TMV, N-like spots were also found, and they were temperature-sensitive. Finally, it was determined that the resistance of tobacco CV58 originated from N gene. 2.CV58 plants were inoculated with TMV or PBSs respectively. The total RNA was extracted and detected by Illumina Hiseq 2000 sequencing system, and then compared with the known database, the differentially expressed miRNAs in two samples of RNA were screened. The differentially expressed miRNA was selected. Divided into up-regulated and down-regulated groups, Target genes were predicted for selected miRNA, go enrichment analysis and KEGG analysis were carried out to predict possible signal pathways. Expression profile analysis showed that among 329 known miRNA with differential expression, there were 211 up-regulated genes. The down-regulation of 118 p0.05 miRNA(Fold-change1.00 and 72 miRNA differentially expressed profiles of 184 Fold-change-1.00 of 256 significantly different miRNA(Fold-change1.00; Informatics analysis of target genes pointing to plant-pathogenic bacteria interaction, cell development and division. There are 7 pathways such as R-mediated phagocytosis of resistance gene, which provide basis for further verification of the role of miRNA in the mechanism of plant antivirus. 3. TMV was used to infect the leaves of tobacco CV58. The stability of 14 candidate internal reference genes including 9 mature miRNAs and 5 traditional housekeeping genes were evaluated by GeNorm and NorFinder programs. The results showed that under the stress of TMV infection, the stability of 14 candidate reference genes in CV58 tissues and leaves of tobacco was evaluated. The most suitable internal reference gene for mature miRNA is L25 miR159, and the most suitable combination is L25 Ntubc2.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.72;Q943.2

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