通过对模板DNA、Primer、dNTPs、10×Taq 10 Buffer、Taq酶的不同浓度进行单因素筛选,并对退火温度和循环次数进行摸索,建立和优化了灰葡萄孢菌Flipper转座子PCR扩增体系。采用该体系及Flipper转座子引物扩增得到了1 159 bp的片段,通过比对发现,该片段与Botryotinia fuckeliana的Flipper转座因子转座酶基因相似性达99%,表明扩增得到的是预期目的片段。Flipper转座子扩增体系的建立,为深入研究我国葡萄灰霉病菌菌株的转座子类型及其与致病力、抗药性等因素之间的关系奠定了重要的基础。
Grape gray mold caused by Botrytic cinerea Pers. is one of the most important diseases on grapes worldwide. The genetic variation of Botrytis cinerea is widely and adaptable,which is easy to produce drug-resistant strains. It is reported that Boty and Flipper transposon of Botrytis cinerea are related to the presence or absence of resistant strains. By setting different gradient and optimize Botrytis cinerea Flipper transposon PCR amplification system of grape in China, including template DNA,primer,dNTPs, 10 × Taq 10 Buffer,Taq DNA polymerase,annealing temperature,cycle indexthe 1 159 bp fragment was obtained ,which has 99% similarity with Botryotinia fuckeliana Flipper transposable element transposase gene through NCBI blastn. So the amplified fragment is verified as expected target fragment. The establishment and optimization of Flipper transposon PCR amplification system of the grape Botrytis cinerea in China provide an important foundation to further study of the relationship between the transposable elements,pathogenicity and resistant
[1] 严红,燕继晔,王忠跃,等. 葡萄灰霉病菌对3 种杀菌剂的多重抗药性检测[J]. 果树学报,2012,29( 4) :625 - 629.
[2] 陈林凤. 新疆灰霉菌遗传多样性及三种生防菌对灰霉菌的拮抗作用初探[D]. 乌鲁木齐: 新疆农业大学,2009.
[3] Giraud T,Fortini D,Levis C, et al. RFLP markers show genetic recombination in Botryotinia fuckeliana ( Botrytis cinerea) and transposable elements reveal two sympatric species[J]. Mol Biol Evol, 1997, 14: 1177 - 1185.
[4] 雷百战,李国英,石在强. 葡萄灰霉病病原鉴定和生物学特性研究[J]. 石河子大学学报: 自然科学版, 2004,22( 增刊) : 145 - 149.
[5] 李喜玲. 灰葡萄孢致病力分化及与胞壁降解酶活性的关系研究[D]. 合肥: 安徽农业大学, 2008.
[6] 张夏兰,李兴红,蔡建波,等. 6 种农药和2 种拮抗菌对葡萄灰霉病的防治试验[J]. 中外葡萄与葡萄酒, 2011( 1) : 27 - 30.
[7] 余文贵,董友磊,赵丽萍,等. 灰霉病菌SSR 遗传多样性分析[J]. 江苏农业科学, 2011, 39( 4) : 18 - 20.
[8] 范咏梅,陈林凤,郝敬喆,等. 新疆灰霉病菌多态性及其致病力分化分析[J]. 中国生态农业学报,2010,18( 3) : 548 - 555.
[9] Esterio M,Muoz G,Ramos C, et al. Characterization of Botrytis cinerea isolates present in Thompson Seedless Table Grapes in the Central Valley of Chile[J]. Plant Dis,2011, 95: 683 - 690.
[10] 董玉霞. 灰葡萄孢除草相关基因的克隆与功能分析[D]. 保定: 河北农业大学, 2008.
[11] Gastón M,Patricio H,Yves B, et al. Genetic characterisation of Botrytis cinerea populations in Chile[J]. The British Mycological Society, 2002, 106( 5) : 594 - 601.
[12] Kálmán Z Váczy,Erzsébet S,Levente K, et al. Sexual recombination in the Botrytis cinerea populations in Hungarian Vineyards[J]. Population Biology,2008,98: 1312 -1319.