發(fā)布時間：2018-03-26 06:51

  本文選題：棉花黃萎病　切入點：大麗輪枝菌　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：棉花黃萎病又被稱為棉花癌癥,其主要致病菌為大麗輪枝菌(Verticillium dahliae)。棉花黃萎病是棉花種植中最嚴(yán)重的病害之一,至今尚無控制該病害的有效方法,主要原因之一在于大麗輪枝菌可產(chǎn)生抗逆性極強的休眠體結(jié)構(gòu),即微菌核,該結(jié)構(gòu)可在土壤中存活數(shù)年以上。關(guān)于大麗輪枝菌致病機理研究相對滯后,目前普遍認(rèn)可的是毒素致萎學(xué)說,該學(xué)說認(rèn)為大麗輪枝菌分泌的毒素是導(dǎo)致寄主產(chǎn)生黃萎癥狀的關(guān)鍵因素。研究發(fā)現(xiàn),毒素引起寄主葉片干枯,黃萎,局部壞死,嚴(yán)重時葉片脫落,甚至整個植株死亡。但目前對毒素的致病機理還缺乏足夠的了解,已有的研究表明,大麗輪枝菌毒素成份復(fù)雜,毒素中單一有效致萎成份尚未被成功分離。微菌核是大麗輪枝菌侵染寄主的初侵染菌源,其形成的數(shù)量和存活情況決定病害發(fā)生程度。有研究表明,微菌核的形成影響大麗輪枝菌致病力。為進(jìn)一步研究微菌核的形成和發(fā)育與其致病力的關(guān)系,本研究以Vd991為材料,利用ATMT方法篩選出不產(chǎn)生微菌核的突變體,通過Southern blot鑒定T-DNA以單拷貝的形式插入突變體的基因組中。利用TAIL-PCR方法確定T-DNA插入突變體CYP52A11基因中。利用RACE技術(shù),獲得CYP52A11基因的cDNA序列,全長為1869bp,其中編碼區(qū)為1548 bp,編碼一個由516個氨基酸組成的蛋白。對比cDNA與DNA序列,確定該基因包含5個外顯子和4個內(nèi)含子,T-DNA插入到第5個外顯子上。對該蛋白進(jìn)行結(jié)構(gòu)域分析顯示,蛋白序列的第1-27位氨基酸區(qū)域為信號肽結(jié)構(gòu)域,說明CYP52A11蛋白有一個跨膜定位的過程,第73-497位氨基酸區(qū)域為P450結(jié)構(gòu)域。利用互補實驗獲得了突變體的功能互補菌株,其表型與野生型相似,有大量微菌核產(chǎn)生。突變體對棉苗的致病性檢測結(jié)果顯示,其對棉苗的致病力下降。通過qRT-PCR方法分析突變體中與微菌核形成相關(guān)基因的表達(dá)情況,與野生型相比,突變體中VDH1, VdPKAC1,VTP1和Cerevisin的表達(dá)量均明顯下降。這些結(jié)果表明,CYP52A11基因在大麗輪枝菌微菌核形成及致病力方面均起重要調(diào)控作用。大麗輪枝菌毒素中有效成份分離是制約其致病機理研究的關(guān)鍵問題之一,毒素中有效致萎成份仍未確定。已有研究表明,毒素中的蛋白組份可能是主要的致萎因子。本研究通過親和層析及凝膠過濾層析對毒素進(jìn)行分離純化,利用棉花檢測純化產(chǎn)物的致病性。結(jié)果顯示,分子量在18.4-25kDa的低分子量蛋白中存在主要致萎成份。選取其中分子量為20kDa的蛋白進(jìn)行質(zhì)譜檢測,結(jié)果經(jīng)Mascot軟件分析,篩選出大麗輪枝菌中有6種蛋白質(zhì)得分為70以上。經(jīng)分析,其中細(xì)胞壁蛋白PhiA可能是一個致病因子。因此,對Vd991中編碼細(xì)胞壁蛋白PhiA的基因進(jìn)行了克隆和原核表達(dá),最終成功表達(dá)出該蛋白。以上實驗結(jié)果表明,大麗輪枝菌CYP52A11基因參與微菌核的形成和發(fā)育,并影響微菌核形成相關(guān)基因的表達(dá)；CYP52A11基因的突變降低了Vd991的致病力,表明微菌核的形成與Vd991的致病力密切相關(guān)。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基因為編碼主要致萎成份的候選基因。這些結(jié)果為揭示大麗輪枝菌的致病機理提供了新的實驗證據(jù),也為最終控制棉花黃萎病的發(fā)生提供了理論依據(jù)。
[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.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.621.2

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