本文選題：灰霉病菌　切入點：農桿菌介導的轉化　出處：《吉林大學》2015年碩士論文　論文類型：學位論文

【摘要】：植物灰霉病是由灰霉病菌（Botrytis cinerea）侵染引起的真菌性病害，在全球廣泛分布，每年造成巨大的經濟損失�；颐共【堑湫偷乃荔w營養(yǎng)型植物病原真菌，在自然條件下多以分生孢子作為侵染寄主的初侵染和再侵染來源�；颐共【菤鈧髦参锊≡�，能侵染200多種寄主植物。自噬相關蛋白9（Atg9）是自噬體（autophagosome）膜形成所必需的一個泛素樣蛋白質，由117個氨基酸殘基組成，負責介導細胞中依賴于溶酶體/液泡等細胞器的生物大分子的周轉。本研究主要闡述Atg9基因在灰霉病菌生長、發(fā)育和致病性中的作用，主要結果如下： 1.以田間灰霉病菌強致病力菌株Bc3-4為原始材料構建了含有3000多個T-DNA隨機插入突變的轉化子群體，采用番茄、草莓離體葉片分別對所獲得的轉化子群體獨立進行至少3次的致病性篩選，共獲得148個侵染表型明顯下降的灰霉病菌T-DNA插入突變體。 2.采用熱不對稱交錯PCR（Thermal Asymmetric Interlaced PCR, TAIL-PCR）技術對部分獲得的侵染表型明顯致弱的灰霉病菌突變體進行T-DNA插入灰霉病菌基因組的遺傳座位分析，發(fā)現(xiàn)其中的一個突變體的T-DNA插入灰霉病菌Atg9基因的啟動子區(qū)域，，Southern blot分析結果顯示該突變是由單拷貝的T-DNA插入造成的。由于很少有關于自噬基因調控灰霉病菌生長發(fā)育及致病性的研究報導，因此，本研究對灰霉病菌Atg9基因進行了相關研究。 3.通過農桿菌介導的遺傳轉化(Agrobacterium tumefaciens mediatedtransformation, ATMT)法成功敲除灰霉病菌的Atg9基因，經過PCR等方法驗證獲得了灰霉病菌Atg9基因的敲除突變體△BcAtg9。 4.通過灰霉病菌野生型Bc05.10與其Atg9基因敲除突變體△BcAtg9菌株生長發(fā)育對比研究發(fā)現(xiàn)：在PDA培養(yǎng)基上，△BcAtg9與野生型菌株生長速率并無明顯的加快或減慢現(xiàn)象，說明Atg9基因不影響菌絲體的生長；產孢量分析顯示野生型每個平板的產孢量約為（3.1±0.4）×107個孢子/plate，而△BcAtg9突變株的產孢量約為（1.5±0.3）×107個孢子/plate，產量約下降一倍，顯著低于野生型菌株分生孢子的產孢量；致病性測定結果表明，在接種3天后，野生型菌株侵染導致的病斑面積為（1.24±0.14）cm2，而突變株侵染導致的病斑面積為（0.34±0.06）cm2，顯著小于野生型菌株侵染導致的病斑面積，說明灰霉病菌△BcAtg9突變株的致病力顯著下降；上述結果與T-DNA插入灰霉病菌Atg9基因啟動子導致突變體致病性下降的結果是一致的。 上述研究結果表明：灰霉病菌Atg9基因不影響其菌絲體的正常生長和分子孢子的形態(tài)形成，但對產孢量和致病性有顯著的影響，表明Atg9基因參與灰霉病菌的致病過程。
[Abstract]:Botrytis cinerea is a fungal disease caused by Botrytis cinerea. It is widely distributed in the world and causes huge economic losses every year. Under natural conditions, conidia are used as the primary and re-infective source of the host. Gray mold is the pathogen of airborne plants. Autophagosome 9 is a ubiquitin like protein necessary for autophagosome membrane formation and consists of 117 amino acid residues. In this study, the role of Atg9 gene in the growth, development and pathogenicity of Botrytis cinerea was studied. 1. A transformation population containing more than 3000 T-DNA insertion mutations was constructed by using the strong pathogenicity strain Bc3-4 in the field, and tomato was used. The isolated leaves of strawberry were screened for pathogenicity at least 3 times, and 148 T-DNA insertion mutants were obtained. 2. The T-DNA insertion genetic loci of the partially acquired mutants with obviously weakened infection phenotype were analyzed by PCR(Thermal Asymmetric Interlaced PCR (TAIL-PCR) technique, which were inserted into the genome of Botrytis cinerea. Southern blot analysis of the promoter region of the Atg9 gene of one of the mutants showed that the mutation was caused by a single copy of T-DNA insertion. There is little evidence that autophagy genes regulate the growth of cinerea cinerea. A study of development and pathogenicity, Therefore, the Atg9 gene of Botrytis cinerea was studied in this study. 3. Agrobacterium tumefaciens mediated transformation (ATMTT) method was used to successfully knockout the Atg9 gene of Botrytis cinerea. The Atg9 gene knockout mutant BcAtg9 was obtained by PCR and other methods. 4. By comparing the growth and development of wild type Bc05.10 and Atg9 knockout mutant BcAtg9, it was found that the growth rate of BcAtg9 and wild-type strain on PDA medium was not significantly accelerated or slowed down. The results showed that Atg9 gene had no effect on the growth of mycelia, the sporulation of wild type was about 3.1 鹵0.4) 脳 107 spore / plateau, while that of BcAtg9 mutant was about 1.5 鹵0.3) 脳 107 spore / plateau, and the yield of wild type was about double. The sporulation of conidia was significantly lower than that of wild-type strains, and the pathogenicity test showed that, after 3 days of inoculation, The spot area caused by wild-type strain infection was 1.24 鹵0.14 cm ~ 2, while that of mutant strain was 0.34 鹵0.06 cm ~ 2, which was significantly smaller than that caused by wild type strain infection, which indicated that the pathogenicity of BcAtg9 mutant was significantly decreased. The above results were consistent with the results of T-DNA insertion of the Atg9 gene promoter of gray mold bacteria resulting in the decrease of pathogenicity of the mutant. The results showed that the Atg9 gene did not affect the normal growth of mycelium and the formation of molecular spores, but had a significant effect on sporulation and pathogenicity, indicating that the Atg9 gene was involved in the pathogenicity of cinerella cinerea.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S432.4

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