猕猴桃抗溃疡病基因的生物信息学分析

发布时间：2018-05-11 10:40

本文选题：猕猴桃 + 生物信息学　；参考：《浙江大学》2016年硕士论文

【摘要】：猕猴桃病害严重影响了猕猴桃的品质与产量,其中由丁香假单胞杆菌猕猴桃致病变种P. Syringae pv. actinidae(PSA)引起的猕猴桃细菌性溃疡病是最重要的病害之一。本研究从猕猴桃的抗病基因入手,分析植物抗病基因的序列结构与其功能的关系,找到能辅助猕猴桃抗溃疡病植株分子育种的标记,结果如下：1.以核苷酸结合位点(Nucleotide binding site, NBS)类基因保守序列,对‘红阳’猕猴桃全基因组中所有96条NBS-LRR类基因,利用Kiwifruit Genome Database、P fam和Coils Server网站,和Bioedit、MEGA 5.0、ClustalW软件,分析确定‘红阳’猕猴桃的NBS-LRR基因类型、结构和系统发育学关系。在‘红阳’猕猴桃全基因组中共有96个NBS-LRR类基因,其中27条NBS-LRR类基因分布在基因簇内,可根据其结构进一步划分为20类基因。并对NBS-LRR类基因家族进行了氨基酸多序列比对和系统进化树分析,发现整个NBS-LRR基因家族可分为三个亚家族。相对其它物种,红阳猕猴桃NBS-LRR类基因总数少,缺少TIR结构,具有LRR结构的基因所占比重低,NBS结构不完整,这些特点可能与其感病性有关联。2.以易感猕猴桃溃疡病品种‘红阳’猕猴桃全基因组中所有数据为基础,寻找到有可能存在猕猴桃抗溃疡病基因的两个抗病基因家族Pto-like基因家族和NBS-LRR基因家族的基因序列,并基于该序列设计引物,以溃疡病抗病品种‘徐香’猕猴桃的全基因组DNA为模板进行PCR扩增,克隆并测序最终得到41条能够通读的不重复序列序列,分析其结构特点并与‘红阳’猕猴桃的NBS-LRR基因序列结构进行对比分析,发现两个品种的NBS-LRR基因中几乎都没有Tir结构域,且NBS结构域结构特点有一定差异：在构成NBS结构的8个结构域中,相比‘红阳’猕猴桃,‘徐香’猕猴桃的NBS-LRR序列都具有更完整的Kinase2结构域,但是缺失RNBS-D结构和MHDVmotif结构比较严重；总体上来说,‘徐香’猕猴桃的NBS结构域更为完整。3.在‘红阳’猕猴桃全基因组序列中国搜集整理得到NBS家族(共96条)和RLK家族(共261条)两大抗病基因家族的序列,应用利用GRAMENE网站在线工具分析其结构,找到68对SSR位点,以此设计和筛选出最终产物在110-340bp,引物长度为18-22bp的引物,以17个品种猕猴桃全基因组DNA为模板扩增,最终筛选得到的两对引物基本能够在17个猕猴桃品种中表现出良好的扩增效果并表现出与溃疡病抗性有一定的相关性。
[Abstract]:The quality and yield of kiwifruit were seriously affected by kiwifruit disease. The bacterial canker of kiwifruit caused by P. Syringae pv. actinidae, a pathogenic variety of Actinidia, is one of the most important diseases. In this study, we analyzed the relationship between the sequence structure and function of disease resistance genes in kiwifruit, and found the markers that can assist molecular breeding of kiwifruit canker resistant plants. The results are as follows: 1. Using the conserved sequence of nucleotide binding site, NBS) class gene at nucleotide binding site, all 96 NBS-LRR class genes in the whole genome of 'Hongyang' kiwifruit were obtained by using Kiwifruit Genome Database P fam and Coils Server website, and BioeditGA-MEGA 5.0 / ClustalW software. The NBS-LRR gene type, structure and phylogenetic relationship of 'Hongyang' kiwifruit were analyzed. There are 96 NBS-LRR class genes in the whole genome of 'Hongyang' kiwifruit, of which 27 NBS-LRR genes are distributed in the gene cluster, which can be further divided into 20 classes according to their structure. The amino acid sequence alignment and phylogenetic tree analysis of NBS-LRR gene family showed that the whole NBS-LRR gene family could be divided into three subfamilies. Compared with other species, the total number of NBS-LRR genes in Actinidia deliciosa is less, the TIR structure is lacking, and the proportion of genes with LRR structure is low. These characteristics may be related to the susceptibility of Actinidia deliciosa. 2. Based on all the data in the whole genome of susceptible kiwifruit canker variety 'Hongyang', the gene sequences of two resistant gene families, Pto-like gene family and NBS-LRR gene family, which may exist in kiwifruit canker resistance genes were found. Based on this sequence, primers were designed to amplify the whole genomic DNA of the disease resistant cultivar 'Xuxiang' kiwifruit by PCR amplification. Finally, 41 non-repeat sequences were cloned and sequenced. It was found that there was almost no Tir domain in the NBS-LRR gene of the two cultivars by analyzing its structural characteristics and comparing it with the sequence structure of the NBS-LRR gene of 'Hongyang' kiwifruit (Actinidia deliciosa). The structural characteristics of NBS domain were different: the NBS-LRR sequences of 'Hongyang' kiwifruit 'Xuxiang' kiwifruit had more complete Kinase2 domain than 'Hongyang' kiwifruit 'Xuxiang' kiwifruit's NBS-LRR domain. In general, the NBS domain of Actinidia chinensis was more complete. 3. Two major resistant gene families of 'Hongyang' kiwifruit ('Hongyang' kiwifruit) were collected and sequenced in China. The sequences of the NBS family (96 genes) and the RLK family (261 genes) were sequenced. Using the GRAMENE website online tools, 68 pairs of SSR loci were found. The primers with the length of 110-340 BP and primer length of 18-22bp were designed and screened. The genomic DNA of 17 varieties of kiwifruit was used as template. The two pairs of primers obtained from the final screening showed good amplification effect in 17 kiwifruit cultivars and showed a certain correlation with the resistance to canker disease.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S436.634.1

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