大丽轮枝菌Vd991致病机理研究

发布时间：2018-03-26 06:51

本文选题：棉花黄萎病　切入点：大丽轮枝菌　出处：《中国农业大学》2016年博士论文

【摘要】：棉花黄萎病又被称为棉花癌症,其主要致病菌为大丽轮枝菌(Verticillium dahliae)。棉花黄萎病是棉花种植中最严重的病害之一,至今尚无控制该病害的有效方法,主要原因之一在于大丽轮枝菌可产生抗逆性极强的休眠体结构,即微菌核,该结构可在土壤中存活数年以上。关于大丽轮枝菌致病机理研究相对滞后,目前普遍认可的是毒素致萎学说,该学说认为大丽轮枝菌分泌的毒素是导致寄主产生黄萎症状的关键因素。研究发现,毒素引起寄主叶片干枯,黄萎,局部坏死,严重时叶片脱落,甚至整个植株死亡。但目前对毒素的致病机理还缺乏足够的了解,已有的研究表明,大丽轮枝菌毒素成份复杂,毒素中单一有效致萎成份尚未被成功分离。微菌核是大丽轮枝菌侵染寄主的初侵染菌源,其形成的数量和存活情况决定病害发生程度。有研究表明,微菌核的形成影响大丽轮枝菌致病力。为进一步研究微菌核的形成和发育与其致病力的关系,本研究以Vd991为材料,利用ATMT方法筛选出不产生微菌核的突变体,通过Southern blot鉴定T-DNA以单拷贝的形式插入突变体的基因组中。利用TAIL-PCR方法确定T-DNA插入突变体CYP52A11基因中。利用RACE技术,获得CYP52A11基因的cDNA序列,全长为1869bp,其中编码区为1548 bp,编码一个由516个氨基酸组成的蛋白。对比cDNA与DNA序列,确定该基因包含5个外显子和4个内含子,T-DNA插入到第5个外显子上。对该蛋白进行结构域分析显示,蛋白序列的第1-27位氨基酸区域为信号肽结构域,说明CYP52A11蛋白有一个跨膜定位的过程,第73-497位氨基酸区域为P450结构域。利用互补实验获得了突变体的功能互补菌株,其表型与野生型相似,有大量微菌核产生。突变体对棉苗的致病性检测结果显示,其对棉苗的致病力下降。通过qRT-PCR方法分析突变体中与微菌核形成相关基因的表达情况,与野生型相比,突变体中VDH1, VdPKAC1,VTP1和Cerevisin的表达量均明显下降。这些结果表明,CYP52A11基因在大丽轮枝菌微菌核形成及致病力方面均起重要调控作用。大丽轮枝菌毒素中有效成份分离是制约其致病机理研究的关键问题之一,毒素中有效致萎成份仍未确定。已有研究表明,毒素中的蛋白组份可能是主要的致萎因子。本研究通过亲和层析及凝胶过滤层析对毒素进行分离纯化,利用棉花检测纯化产物的致病性。结果显示,分子量在18.4-25kDa的低分子量蛋白中存在主要致萎成份。选取其中分子量为20kDa的蛋白进行质谱检测,结果经Mascot软件分析,筛选出大丽轮枝菌中有6种蛋白质得分为70以上。经分析,其中细胞壁蛋白PhiA可能是一个致病因子。因此,对Vd991中编码细胞壁蛋白PhiA的基因进行了克隆和原核表达,最终成功表达出该蛋白。以上实验结果表明,大丽轮枝菌CYP52A11基因参与微菌核的形成和发育,并影响微菌核形成相关基因的表达；CYP52A11基因的突变降低了Vd991的致病力,表明微菌核的形成与Vd991的致病力密切相关。此外,Vd991毒素成份中低分子量非糖蛋白是主要致萎成份,其中,PhiA基因为编码主要致萎成份的候选基因。这些结果为揭示大丽轮枝菌的致病机理提供了新的实验证据,也为最终控制棉花黄萎病的发生提供了理论依据。
[Abstract]:Cotton Verticillium wilt is also known as cotton cancer, the main pathogenic bacteria to Verticillium dahliae (Verticillium dahliae). Cotton Verticillium wilt is one of the most serious diseases of cotton planting, there is no effective method to control the disease, one of the main reasons is that V.dahliae can sleep structure produce strong resistance, i.e. microsclerotia in soil, the structure can survive for several years. On Verticillium dahliae pathogenicity research lagged behind, now generally accepted is the toxin wilting doctrine, the doctrine that Verticillium dahliae toxin is the key factor leading to host produce Verticillium wilt symptoms. The study found that toxin induced host leaves dry, yellowing, necrosis, leaf abscission seriously, even the whole plant death. But the pathogenic mechanism of toxin is lack of sufficient understanding of the existing research shows that the toxin of Verticillium dahliae ingredient complex Miscellaneous, single effective wilting toxin composition has not been successfully isolated. Microsclerotia of Verticillium dahliae infecting the host is the primary infectious pathogen, disease degree decided its formation quantity and survival. Studies have shown that the formation of microsclerotia of Verticillium dahliae pathogenicity. For further study of relation between the formation of microsclerotia and development and pathogenicity, on the basis of Vd991 material, selected by ATMT method does not produce microsclerotia by Southern blot identification of T-DNA mutants with single copy insertion mutant forms of the genome. Determine the T-DNA insertion mutants of CYP52A11 gene by using TAIL-PCR method. Using RACE technology, cDNA sequence the CYP52A11 gene was 1869bp, encoding, which is 1548 BP, encoding a protein with 516 amino acids. Sequence comparison of cDNA and DNA, the gene contains 5 exons and 4 introns T-DNA, inserted in the fifth exon. Domain analysis showed that the protein, a 1-27 amino acid region of protein sequence of signal peptide domain, which indicated that the CYP52A11 protein had a transmembrane localization process, the 73-497 amino acid region P450 domain. By using the obtained mutant complementation experiments functional complementation strains, the phenotype is similar to the wild type, there are a large number of microsclerotia. Mutant of cotton seedling pathogenicity test results showed that the decline of cotton seedling pathogenicity. Through the qRT-PCR analysis of the expression of related genes and microsclerotia formation in the mutant compared with the wild-type, mutant VDH1, VdPKAC1. The expression of VTP1 and Cerevisin were significantly decreased. These results suggest that CYP52A11 gene in Verticillium dahliae microsclerotia formation and pathogenicity plays an important role in regulation. Effective separation of Verticillium dahliae toxin Is one of the key problems restricting the pathogenic mechanism of the toxin in effective wilting composition remains uncertain. Studies have shown that the toxin protein component is wilting factor mainly. Through the study of the toxin was purified by affinity chromatography and gel filtration chromatography, purified by pathogenic detection. The results showed that cotton the molecular weight, there are wilting ingredients in the low molecular weight protein 18.4-25kDa. The molecular weight of 20kDa protein by mass spectrometry, the results analyzed by Mascot software, screening of Verticillium dahliae in 6 proteins score above 70. After analysis, the cell wall protein PhiA may be a pathogenic factor. Therefore, the encoding cell wall protein PhiA in Vd991 gene by cloning and prokaryotic expression, finally successfully expressed the protein. These results indicate that Verticillium dahliae CYP52A11 gene. With the formation and development of microsclerotia, and influence the expression of microsclerotia formation related genes; CYP52A11 mutation reduced the pathogenicity of Vd991 and Vd991, indicating the formation of microsclerotia of pathogenicity is closely related. In addition, Vd991 toxin components of low molecular weight glycoprotein is mainly non wilting ingredients, the PhiA gene as a candidate the gene encoding the main wilting component. These results provide new experimental evidence to reveal the pathogenic mechanism of Verticillium dahliae, but also provides a theoretical basis for the eventual control of Cotton Verticillium wilt.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.621.2

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