马铃薯晚疫病菌四个RxLR基因的功能鉴定

发布时间：2018-03-04 01:18

本文选题：马铃薯晚疫菌　切入点：龙葵　出处：《山东农业大学》2016年博士论文　论文类型：学位论文

【摘要】：马铃薯晚疫病是由致病疫霉菌(Phytophthora infestans(Mont.)de Bary)引起,能够导致马铃薯的大幅减产,甚至绝收,严重威胁着马铃薯生产,被视为国际第一大作物病害。其病原菌在与植物协同进化的过程中,会分泌丰富的Rx LR类效应分子,发挥了不同的致病作用,成为侵染的“武器”;同时,植物免疫系统进行防卫反应形成“盾”,目前鉴定的抗晚疫病基因,其识别的无毒基因也都属于病原菌Rx LR类分泌蛋白。由于马铃薯栽培品种是四倍体作物,自交不亲和,通过杂交的方法将野生种中鉴定的抗病基因导入到栽培品种从而获得抗病效果的策略只能在有限的资源中利用。因此,从野生种质资源中鉴定抗晚疫病基因并利用基因工程技术培育转基因抗病马铃薯品种成为重要的防治策略之一。龙葵作为马铃薯晚疫病非寄主茄科植物,对马铃薯晚疫病菌具有很好的抗性,预测携带相应的抗病基因资源。本研究通过构建我国A1融合群晚疫病黑龙江分离菌株HLJ1的Rx LR蛋白基因池,进而在非寄主龙葵上筛选候选无毒基因并进行分析,帮助解释非寄主抗性的遗传基础并后续帮助从龙葵中克隆抗病基因。目前,取得的研究成果包括以下几点:1.参考已测序菌株T30-4基因组序列,以HLJ1菌株的gDNA为模板,通过高保真PCR,成功克隆并构建251个Rx LR效应因子的瞬时表达载体。利用生物信息学手段分析两个菌株T30-4和HLJ1基因组中Rx LR分子的多态性,除了8个基因在部分菌株中存在等位基因缺失外,其它Rx LR效应分子存在对应的等位基因。等位基因之间编码氨基酸序列完全相同数量相对较多,部分基因存在不同数量的多态性。2.为研究Rx LR效应因子的功能,利用PVX介导的瞬时表达系统将构建的Rx LR效应子分别在本氏烟和龙葵上进行瞬时表达,筛选得到3个仅能在龙葵上触发过敏性坏死(HR)的基因,作为候选抗性无毒基因(Pi15718、Pi15556、Pi21190)。通过荧光定量分析发现这三个基因在HLJ1侵染过程中表达量都出现明显变化。通过对这3个Rx LR效应分子的功能结构域的截短分析发现,确定其与龙葵识别产生HR的关键区域都在C-端。3.进一步解析Pi15718的功能,对其在10个晚疫菌株中的多态性进行分析发现存在两种类型的氨基酸序列,其中Pi15718HLJ1类可以在除龙葵品种(SN005)外的其他测试的龙葵上均产生HR,Pi1571888069类不能在龙葵上产生HR。推测氨基酸改变影响其在龙葵上产生HR的功能。有意思的是,Pi1571888069和Pi15718HLJ1的C末端单独可以激发龙葵的HR,暗示Pi1571888069N-端具有抑制C-端激活龙葵HR的功能。酵母双杂交验证N-端并不与C-端发生蛋白的互作。N-端抑制功能的关键位置确定在Pi1571888069的第45-52位的氨基酸之间。同时,为研究该候选无毒基因的功能,稳定的转基因株系以及酵母双杂交筛选互作蛋白的工作也在进行中。4.针对候选无毒基因Pi15556,研究发现在15个晚疫菌株中高度保守,氨基酸序列完全一致。其在供测的龙葵样本SN004、SN005、SN008中也不激发HR,体现无毒基因的特征。为了研究其在菌株中的功能,构建Pi15556的抑制突变体菌株,在鉴定了突变率分别为61.9%和34.7%的突变体M-1和M-2。对突变体进行生物学分析发现Pi15556并不影响菌落形态,菌丝生长和孢子囊形态,但预测可能通过影响产孢能力影响致病性。5.Pi21190也有类似结果,在不同的龙葵材料中分别可激发或者不激发HR反应,体现无毒基因的特征。同时,其在不同菌株中存在不同的等位基因,但其等位基因在龙葵SN0022上均产生HR,体现较强的保守性。6.同期筛选到一个可以在本氏烟上抑制INF1产生HR的Rx LR效应分子Pi14684进行等位基因的多态性分析,发现其存在多个氨基酸位点的改变,在本氏烟上瞬时表达结果证明E43K,是其抑制INF1产生HR的关键位点。随后为了研究Pi14684是否特异性的抑制INF1产生HR,分别接种SFI2,Avr3aKI和R3a混合菌液,发现Pi14684仅能特异性抑制INF1介导的HR。
[Abstract]:Potato late blight caused by Phytophthora infestans (Phytophthora infestans (Mont.) de Bary) can lead to a substantial reduction, caused by potato crops, a serious threat to potato production, is regarded as the first major international crop diseases. The pathogenic bacteria in the co evolutionary process of plants, will produce rich Rx LR effect molecules play a pathogenic role in different infection, become "weapon"; at the same time, the immune system of plant defense response form a "shield", the identification of late blight resistance gene, its avirulence gene recognition also belong to the pathogen Rx LR secreted protein. Because the potato cultivars are tetraploid crops, self incompatibility and through the hybrid approach will only identify wild species in the strategy of resistance genes into cultivars to gain resistance effect by making use of the limited resources. Therefore, from wild germplasm identification Set late blight resistance gene by gene engineering technology and cultivation of disease resistant transgenic potato varieties has become one of the important control strategy. As the potato late blight of Solanum nigrum non host plants of Solanaceae, has good resistance to potato late blight pathogen, resistance gene prediction carrying the corresponding resources. This paper constructed the Rx LR protein gene pool in China A1 the fusion group of late blight in Heilongjiang isolates of HLJ1, then the candidate avirulence genes in non host selection of Solanum nigrum and analyzed to help explain the genetic basis of non host resistance and subsequent help resistance gene cloning from Solanum nigrum. At present, the research achievements include the following: 1. reference sequenced strain T30-4 genome sequence in HLJ1 strain gDNA as template by high fidelity PCR, transient expression was successfully cloned and constructed 251 Rx LR effect factor of the carrier. By the method of bioinformatics analysis two The polymorphism of Rx LR strains T30-4 and HLJ1 molecules in the genome, in addition to the 8 gene deletion allele in some strains, there are corresponding alleles of other Rx molecules. The LR effect between the allele encoding amino acid sequence identical to a relatively large number of parts gene polymorphism of.2. has a different number of for the study of Rx LR effect factor, Rx LR effect will be constructed by PVX mediated transient expression system respectively in Nicotiana benthamiana and Solanum nigrum on transient expression, screened 3 can only trigger hypersensitive necrosis in Solanum nigrum on (HR) gene as candidate resistance avirulence genes (Pi15718, Pi15556, Pi21190). By fluorescence quantitative analysis found that the expression level of three genes in the HLJ1 infection process are significant changes. By truncating the functional domains of the 3 Rx LR effect of molecular analysis, and determine its knowledge of Solanum nigrum Don't have the key region of the HR C-.3. in the end further analysis the function of Pi15718, the 10 strains of late blight in polymorphism analysis shows that there are two types of amino acid sequence, the Pi15718HLJ1 class can be in addition to Solanum nigrum (SN005) and other varieties tested Solanum were HR, Pi1571888069 can not produce in Solanum nigrum HR. guesses amino acid change and its impact on the function of HR in Solanum nigrum. Interestingly, the C terminal Pi1571888069 and Pi15718HLJ1 alone can stimulate HR nigrum can inhibit C-, suggesting that Pi1571888069N- end end activation function of HR. The Dragon Kui the key position of the yeast two hybrid N- end and C- end verification does not occur protein interaction.N- end inhibition function is determined between Pi1571888069 No. 45-52 amino acid. At the same time, to study the candidate avirulence gene function, stable transgenic lines and yeast two hybrid Screening the protein interaction work is underway for the candidate.4. avirulence gene Pi15556, the study found that highly conserved in 15 strains of late blight, identical amino acid sequence. The SN005 in the test sample of SN004, SN008 in Solanum nigrum, did not stimulate HR, reflect the characteristics of avirulence genes. In order to study the strain the function of inhibition of mutant strain Pi15556 was constructed and the mutation rate were 61.9% and 34.7% of the mutants of M-1 and M-2. on the biological analysis found that the Pi15556 mutant did not affect colony morphology identification, mycelial growth and spore morphology, but the prediction by sporulation ability affect the pathogenicity of.5.Pi21190 have similar results in different the materials respectively can stimulate or inspire HR reaction, reflect the characteristics of avirulence genes. At the same time, the different strains have different alleles, but the allele SN00 in Solanum nigrum 22 have HR, reflects the conservative.6. strong earlier screening to a polymorphic Rx LR effector Pi14684 INF1 can inhibit the production of HR in n.benthamiana the allele, found that the existence of multiple amino acid changes, transient expression showed that E43K in n.benthamiana, is the key the site of inhibition of INF1 induced HR. Then in order to investigate whether Pi14684 inhibition of INF1 specific HR, Avr3aKI and R3a were inoculated with SFI2, mixed bacteria, found that Pi14684 only can specifically inhibit INF1 mediated HR.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.32

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