【作者】 刘文婷；

【导师】 吴云锋；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2016， 硕士

【摘要】 小麦蓝矮植原体病害主要发生在小麦上,其常发地集中在我国陕西、山西、内蒙古、宁夏和甘肃等西北地区,这种病害严重的威胁了小麦的生长,造成产量和质量的严重损失。近几年来,小麦蓝矮植原体开展的研究工作主要是围绕其全基因组的测序及致病机制的探索。通过生物信息学软件对小麦蓝矮植原体基因组序列进行分析比较,预测到525个与编码蛋白相关的基因,其中大部分与其他植原体编码蛋白的基因同源,但是也有22种比较特异的基因,同时也分析预测到37个分泌蛋白,并进行了致病性的鉴定,发现SWP1侵染的本氏烟会表现出植株底部分枝增多的症状。本论文针对SWP1进行相关的生物信息学分析并构建不同的关于SWP1的缺失突变体,旨在明确能够引起本氏烟出现底部分枝增多现象的关键区域。具体的实验结果如下:1.对SWP1基因编码蛋白的基本生物学特性和结构进行分析预测:SWP1基因编码的蛋白大小为13.292kDa,等电点pI为9.2,属于碱性蛋白,信号肽的切割位点在SWP1蛋白N端的第33与第34个氨基酸之间,成熟以后分泌到胞外去除信号肽的蛋白大小为9.516 kDa。通过PSIPRED在线软件分析SWP1蛋白的二级结构,结果表明SWP1蛋白主要是在整个氨基酸序列的6-24、48-65、68-79、82-99处形成四个α螺旋。同时,分析预测出在整个氨基酸序列的第82-101个氨基酸处形成一个卷曲螺旋结构并在C末端的存在一个单分型的核定位信号。2.以SWP1生物信息学的结果为基础,构建了SWP1△NLS、SWP1△NLSCC、SWP1△CC、SWP1△N、SWP1△NC这五个缺失突变体,并利用农杆菌介导的植物表达载体在本氏烟中进行表达,通过对本氏烟的症状进行分析,发现缺失了SWP1 C端的卷曲螺旋结构,本氏烟将不再表现出丛枝的症状,而缺失SWP1 C端包含核定位信号在内的10个氨基酸及N端的14个氨基酸并不会影响SWP1功能的发挥,因此,推断SWP1C端的卷曲螺旋结构是其发挥功能的关键。3.在明确了卷曲螺旋结构是SWP1发挥功能的关键部位后,进一步构建了SWP1△NC1、SWP1△NC2、SWP1△NNLSCC这三个缺失突变体,并在本氏烟中进行表达,结果显示缺失卷曲螺旋结构中的第11-15个氨基酸,本氏烟将不会出现分枝增多的症状,因此,推断这5个氨基酸是卷曲螺旋结构中关键的活性区域,同时也是SWP1发挥功能的关键区域。更多还原

【Abstract】 Wheat blue dwarf is one of the main important wheat diseases which occur in rid and semi-arid areas of northwest China, including Shaanxi, Shanxi, Gansu, Ningxia and Inner Mongolia. It imperiled the growth of wheat and significantly influenced the production and quality.In recent years, the research about WBD was primarily focused on fully genome sequencing and pathogenicity. Using the bioinformatics software, 525 protein-coding genes were identified in WBD genome. Except 22 WBD-specific genes, most of them were homologous to genes from other phytoplasma. In the meanwhile, 37 genes encoding for WBD secreted proteins were constructed into pGR107. Thses proteins were expressed in Nicotiana benthamiana by Agrobacterium-mediated viral vector assays. When SWP1 infect Nicotiana benthamiana, the base of the plant stem was manifested as proliferation. In this study, the SWP1 gene of WBD was analysed using bioinformatics. On this basis, different mutants of SWP1 genes were amplified and constructed into pGR107. In order to identify the essential domain of secreted protein SWP1 that caused the symptom of N. benthamiana, these different genes were expressed in Nicotiana benthamiana. The major results are listed as follows:1. Molecular structure characteristics of the SWP1 gene: The gene was deduced to encode a peptide of 112 amino acids whose predicted molecular mass was 13.292 kDa and isoelectric point was 9.2. The signal peptide cleavage site was between the 33 th and the 34 th amino acid at N-terminals of the SWP1 protein, so the SWP1 mature protein had only 80 amino acids and its predicted molecular mass was 9.516 kDa. It was predicted that the secondary structure of SWP1 was constructed by four α-helix folds. The coiled coil structure was between the 82 th to 101 th amino acids at C-terminals. There was a monopartite NLS located in the C terminus of SWP1. Otherwise, homologous modeling was predicted using the swiss-model.2. Based on the results of the bioinformatics, a series of truncation mutants were constructed into pGR107, including SWP1△NLS 、 SWP1△NLSCC 、 SWP1△CC 、 SWP1△N、 SWP1△ NC. These different genes were expressed in Nicotiana benthamiana by Agrobacterium-mediated viral vector assays. Analysis the symptoms of different Nicotiana benthamiana, the results indicated that the ten amino acids at C terminus including the monopartite NLS and the fourteen amino acids at N-terminals are not essential for proliferation, and the coiled coil structure is critical for proliferation of SWP1.3. After identification the coiled coil structure is essential for proliferation, another three truncation mutants were constructed, including SWP1△NC1 、 SWP1△NC2、 SWP1△ NNLSCC. These genes were expressed in Nicotiana benthamiana, the results showed that the 11 th to 15 th amino acids of coiled coil structure were critical for proliferation. This indicated that these five amino acids were essential for coiled coil structure and also critical for proliferation of SWP1.更多还原