本文選題：馬鈴薯晚疫菌　切入點：龍葵　出處：《山東農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：馬鈴薯晚疫病是由致病疫霉菌(Phytophthora infestans(Mont.)de Bary)引起,能夠?qū)е埋R鈴薯的大幅減產(chǎn),甚至絕收,嚴重威脅著馬鈴薯生產(chǎn),被視為國際第一大作物病害。其病原菌在與植物協(xié)同進化的過程中,會分泌豐富的Rx LR類效應(yīng)分子,發(fā)揮了不同的致病作用,成為侵染的“武器”;同時,植物免疫系統(tǒng)進行防衛(wèi)反應(yīng)形成“盾”,目前鑒定的抗晚疫病基因,其識別的無毒基因也都屬于病原菌Rx LR類分泌蛋白。由于馬鈴薯栽培品種是四倍體作物,自交不親和,通過雜交的方法將野生種中鑒定的抗病基因?qū)氲皆耘嗥贩N從而獲得抗病效果的策略只能在有限的資源中利用。因此,從野生種質(zhì)資源中鑒定抗晚疫病基因并利用基因工程技術(shù)培育轉(zhuǎn)基因抗病馬鈴薯品種成為重要的防治策略之一。龍葵作為馬鈴薯晚疫病非寄主茄科植物,對馬鈴薯晚疫病菌具有很好的抗性,預(yù)測攜帶相應(yīng)的抗病基因資源。本研究通過構(gòu)建我國A1融合群晚疫病黑龍江分離菌株HLJ1的Rx LR蛋白基因池,進而在非寄主龍葵上篩選候選無毒基因并進行分析,幫助解釋非寄主抗性的遺傳基礎(chǔ)并后續(xù)幫助從龍葵中克隆抗病基因。目前,取得的研究成果包括以下幾點:1.參考已測序菌株T30-4基因組序列,以HLJ1菌株的gDNA為模板,通過高保真PCR,成功克隆并構(gòu)建251個Rx LR效應(yīng)因子的瞬時表達載體。利用生物信息學(xué)手段分析兩個菌株T30-4和HLJ1基因組中Rx LR分子的多態(tài)性,除了8個基因在部分菌株中存在等位基因缺失外,其它Rx LR效應(yīng)分子存在對應(yīng)的等位基因。等位基因之間編碼氨基酸序列完全相同數(shù)量相對較多,部分基因存在不同數(shù)量的多態(tài)性。2.為研究Rx LR效應(yīng)因子的功能,利用PVX介導(dǎo)的瞬時表達系統(tǒng)將構(gòu)建的Rx LR效應(yīng)子分別在本氏煙和龍葵上進行瞬時表達,篩選得到3個僅能在龍葵上觸發(fā)過敏性壞死(HR)的基因,作為候選抗性無毒基因(Pi15718、Pi15556、Pi21190)。通過熒光定量分析發(fā)現(xiàn)這三個基因在HLJ1侵染過程中表達量都出現(xiàn)明顯變化。通過對這3個Rx LR效應(yīng)分子的功能結(jié)構(gòu)域的截短分析發(fā)現(xiàn),確定其與龍葵識別產(chǎn)生HR的關(guān)鍵區(qū)域都在C-端。3.進一步解析Pi15718的功能,對其在10個晚疫菌株中的多態(tài)性進行分析發(fā)現(xiàn)存在兩種類型的氨基酸序列,其中Pi15718HLJ1類可以在除龍葵品種(SN005)外的其他測試的龍葵上均產(chǎn)生HR,Pi1571888069類不能在龍葵上產(chǎn)生HR。推測氨基酸改變影響其在龍葵上產(chǎn)生HR的功能。有意思的是,Pi1571888069和Pi15718HLJ1的C末端單獨可以激發(fā)龍葵的HR,暗示Pi1571888069N-端具有抑制C-端激活龍葵HR的功能。酵母雙雜交驗證N-端并不與C-端發(fā)生蛋白的互作。N-端抑制功能的關(guān)鍵位置確定在Pi1571888069的第45-52位的氨基酸之間。同時,為研究該候選無毒基因的功能,穩(wěn)定的轉(zhuǎn)基因株系以及酵母雙雜交篩選互作蛋白的工作也在進行中。4.針對候選無毒基因Pi15556,研究發(fā)現(xiàn)在15個晚疫菌株中高度保守,氨基酸序列完全一致。其在供測的龍葵樣本SN004、SN005、SN008中也不激發(fā)HR,體現(xiàn)無毒基因的特征。為了研究其在菌株中的功能,構(gòu)建Pi15556的抑制突變體菌株,在鑒定了突變率分別為61.9%和34.7%的突變體M-1和M-2。對突變體進行生物學(xué)分析發(fā)現(xiàn)Pi15556并不影響菌落形態(tài),菌絲生長和孢子囊形態(tài),但預(yù)測可能通過影響產(chǎn)孢能力影響致病性。5.Pi21190也有類似結(jié)果,在不同的龍葵材料中分別可激發(fā)或者不激發(fā)HR反應(yīng),體現(xiàn)無毒基因的特征。同時,其在不同菌株中存在不同的等位基因,但其等位基因在龍葵SN0022上均產(chǎn)生HR,體現(xiàn)較強的保守性。6.同期篩選到一個可以在本氏煙上抑制INF1產(chǎn)生HR的Rx LR效應(yīng)分子Pi14684進行等位基因的多態(tài)性分析,發(fā)現(xiàn)其存在多個氨基酸位點的改變,在本氏煙上瞬時表達結(jié)果證明E43K,是其抑制INF1產(chǎn)生HR的關(guān)鍵位點。隨后為了研究Pi14684是否特異性的抑制INF1產(chǎn)生HR,分別接種SFI2,Avr3aKI和R3a混合菌液,發(fā)現(xiàn)Pi14684僅能特異性抑制INF1介導(dǎo)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.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.32

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