核桃PR1与PR4基因的克隆及表达分析

发布时间：2018-03-23 05:32

本文选题：核桃　切入点：PR基因　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：核桃(Juglans regia)是胡桃科核桃属落叶落果乔木,属于多年生的经济树木。作为世界四大坚果之一,其核仁具有极其丰富的营养如富含优质的蛋白,多种矿质元素等营养物质,脂肪含量高,素有“木本油料之王”的称号。核桃种植面积在不断扩大,我国已成为世界第一种植大国,2012年核桃种植面积约为42.5万公顷,产量约为170万吨(联合国粮农组织FAO),但近年来,环境变化及病虫害的影响对核桃的生产和经济效益造成的巨大的损失,其中核桃细菌性黑斑病是细菌侵染引起的病害,又称黑斑病,该病害广泛分布在全世界所有主要的核桃产区,严重危害核桃的生产,病程相关蛋白(PRP)是重要的抗病响应蛋白,但在核桃中的鲜有报道。本研究以“香玲”核桃为研究对象,从核桃中克隆了PR1和PR4基因,利用生物信息学分析的方法比较了PR1和PR4基因,通过实时荧光定量技术,比较了两个基因在不同核桃品种冬芽中的表达水平,并分析了细菌性黑斑病病原菌侵染过程中PR1及PR4的相对表达量。本研究的主要结果如下:(1)通过T-A克隆得到核桃的PR1及PR4基因并测序确认了序列,通过BLAST、进化分析及多序比对分析,对这些蛋白的序列特征进行了初步分析。得到核桃的PRI基因与水稻、小麦的PR1基因同源性较高,核桃PR4基因与葡萄的PR4基因同源性较高。(2)核桃细菌性黑斑病严重危害核桃生产,细菌在冬季时潜伏在核桃越冬芽中,对PR1和PR4基因在不同核桃品种中的冬芽表达进行分析发现,PR1在西扶1、辽3、香玲及林3以冬芽中的表达呈现上调表达,而在契可和辽4中表达水平低于对照,呈抑制状态;PR4在西扶1、辽3、香玲、林3以及契可的越冬芽中呈现上调表达,在辽4冬芽中的表达呈抑制状态,表明不同品种核桃PR1及基因中的表达不同,进而预测不同品种的核桃抗病性不同。(3)采用针刺法接种方式对香玲及清香核桃叶片进行细菌性黑斑病病原菌的接种,对病原菌胁迫下的不同品种的核桃叶片进行抗逆性分析,结果表明PR1和PR4在不同品种的核桃接种病原菌后的表达量变化有很大差异,两个基因的相对表达量在清香和香玲中的变化趋势都呈现先上升后下降的趋势,并且在清香中基因的相对表达量明显高于香玲中的相对表达量。为进一步研究PR1和PR4在核桃中的功能奠定了基础。
[Abstract]:Juglans regia (Juglans regia) is a deciduous and fruit-falling tree of the genus Juglans, which is a perennial economic tree. As one of the four largest nuts in the world, its nucleoli are rich in nutrients such as high quality protein, many mineral elements and so on. High fat content, known as the "king of woody oil," walnut growing area is constantly expanding, China has become the world's largest growing country, walnut planting area of about 425000 hectares in 2012, The yield is about 1.7 million tons (FAO), but in recent years, environmental changes and the effects of pests and diseases have caused huge losses to the production and economic benefits of walnut, among which the bacterial black spot of walnut is the disease caused by bacterial infection. Also known as black spot disease, the disease is widely distributed in all the major walnut production areas in the world, seriously endangering the production of walnut, disease course related protein (PRP) is an important disease resistance response protein. In this study, PR1 and PR4 genes were cloned from walnut and PR1 and PR4 genes were compared by bioinformatics. The expression levels of two genes in winter bud of different walnut varieties were compared. The relative expression of PR1 and PR4 in bacterial black spot pathogen was analyzed. The main results were as follows: 1) the PR1 and PR4 genes of walnut were cloned by T-A and sequenced. The sequence characteristics of these proteins were preliminarily analyzed by BLAST, evolutionary analysis and multiple sequence alignment analysis. The results showed that the PRI gene of walnut and the PR1 gene of rice and wheat had high homology. The PR4 gene of walnut and the PR4 gene of grape have high homology. 2) the bacterial black spot of walnut seriously harms the production of walnut, and the bacteria lurk in the bud of walnut overwintering in winter. The expression of PR1 and PR4 genes in winter buds of different walnut varieties was analyzed. It was found that the expression of PR1 and PR4 genes in winter buds of Xinfu 1, Liao3, Xiangling and Lin3 was up-regulated, but the expression level in Qike and Liao4 was lower than that in control. The expression of PR4 was up-regulated in the overwintering buds of Xifu 1, Liao3, Xiangling, Lin3 and Chico, but inhibited in the winter buds of Liao4, indicating that the expression of PR1 and genes in different walnut varieties were different. Then it was predicted that different varieties of walnut had different disease resistance. (3) inoculation method of needle method was used to inoculate the pathogen of bacterial black spot disease in leaves of Xiangling and Flavor walnut, and the resistance of different varieties of walnut leaves under pathogen stress was analyzed. The results showed that the expression of PR1 and PR4 in different varieties of walnut were different after inoculation with pathogen, and the relative expression of the two genes in Flavor and Xiangling showed a trend of first rising and then decreasing. The relative expression of genes in Flavor was significantly higher than that in Xiangling, which laid a foundation for further study on the function of PR1 and PR4 in walnut.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S664.1

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