胶孢炭疽菌致病性减弱突变菌株H800T-DNA插入位点侧翼基因的克隆  

Molecular Cloning of T-DNA Integration Flanking Sequence of Colletotrichum gloeosporioides Pathogenicity-weakened Mutant H800

作　　者：李秋洁[1,2,3] 吴伟怀[2] 贺春萍[2] 郑金龙[2] 习金根[2] 梁艳琼[2] 许沛冬 郑肖兰[2] 易克贤[2,3] 

机构地区：[1]海南大学环境与植物保护学院,海南海口570228 [2]中国热带农业科学院环境与植物保护研究所,海南海口571101 [3]中国热带农业科学院热带生物技术研究所,海南海口571101

出　　处：《热带作物学报》2015年第5期949-955,共7页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金(No.31072076;No.31101408);公益性行业科研专项(No.201303057);中央级公益性科研院所基本科研业务专项(No.2015hzs1J002)

摘　　要：对农杆菌介导转化引起的致病性减弱突变菌株H800的菌落形态、孢子形态、菌丝体生长速率、孢子萌发率和致病力等生物学特性进行研究,并利用TAIL-PCR方法克隆T-DNA插入位点基因,结合生物信息学分析该基因功能与H800表型变化及致病性减弱的关系。结果表明,突变菌株H800的致病力低于野生型菌株CH008;生长速率为0.83 cm/d,亦显著低于CH008的1.13 cm/d;产孢量和分生孢子大小与CH008无明显差异;孢子萌发率为2.58%,显著低于野生型的95.98%;序列分析显示,T-DNA已插入St Cg800基因的启动子区域,基因结构分析表明,St Cg800基因编码区全长774 bp,无内含子,可编码257个氨基酸多肽的St Cg800蛋白。该蛋白是不稳定的水溶性蛋白,亚细胞定位于细胞质,且无信号肽,推测为生长因子类的酶,但功能尚未知。Stylosanthes anthracnose, cuased by Colletotrichum gloeosporioides, is the main disease influencing the production and extension of Stylosanthes. A mutant H800 strain was obtained by Agrobacterium-mediated transformation（ ATMT） method, its pathogenicity to Stylosanthes was weakened. The colony morphology, conidial morphology, mycelial growth rate, conidial germination and pathogenicity of H800 were determined, and Tail-PCR was used to clone the flaking sequence. Additionally, the function of T-DNA insertion region in the genome of H800 was predicted by bioinformatics. Compared to the wide-type strain CH008, H800＇s pathogenicity was significantly declined. The growth rate was 0.83 cm/d, significantly slower than CH800＇s 1.13 cm/d, but the conidiation and the conidia size had no significant difference, the germination rate was 2.58%, which was significantly less than the CH008＇s 95.98%. The flanking sequence was cloned by Tail-PCR, sequence alignment analysis showed that the T-DNA inserted into the promoter region of St Cg800 gene. The gene structural analysis indicated that St Cg800 gene was 774 bp, included only one exon, no intron, encoded a 257 amino acid protein,which was a hypothetical and unstable water-soluble protein, located in cytoplasm, no signal peptide, belonged to growth factor and enzyme.

关 键 词：胶孢炭疽菌 致病性 表型分析 基因克隆 

分 类 号：S435.4[农业科学—农业昆虫与害虫防治]