棉花黄萎病菌T-DNA插入突变体表型特征和侧翼序列分析

Analysis of T-DNA Insertional Flanking Sequence and Mutant Phenotypic Characteristics in Verticillium Dahliae

【目的】为鉴定棉花黄萎病菌随机插入突变体的表型特征,构建黄萎病菌遗传转化和功能基因研究的技术平台。【方法】采用农杆菌介导的T-DNA插入和高效TAIL-PCR方法。【结果】获得了2628个棉花黄萎病菌VD991的T-DNA随机插入的突变体和15个突变体插入位点的侧翼序列。部分突变体的表型特征和插入位点侧翼序列分析表明,①突变体的菌落形态分为菌丝型、菌核型和中间型3类,菌核型约占7.3%;②在摇瓶培养条件下,突变体产孢高峰在接种后第5—6天,菌核型突变体的产孢能力普遍高于菌丝型;③棉花黄萎病菌VD991可以产生胞外蛋白酶、淀粉酶、纤维素酶和果胶酶。筛选获得蛋白酶分泌缺失的突变体3个,淀粉酶分泌缺失的突变体1个,果胶酶分泌降低的突变体6个;④菌核型突变体的致病力普遍高于野生型。获得了6个致病力减弱的突变体;⑤突变体侧翼序列与同种的大丽轮枝菌菌株VDLs.17基因组的序列一致性在95%—100%之间,与不同种的黑白轮枝菌VaMs.102序列的一致性为87%—94%。【结论】农杆菌介导的T-DNA随机插入可用于棉花黄萎病菌突变体的构建,突变体微菌核的产生与其产孢能力、致病性存在相关关系。美国大丽轮枝菌菌株VDLs.17基因组可用作黄萎病菌VD991功能基因研究的参考序列。

[Objective] The objective of the experiment is to identify the phenotype characteristics of T-DNA insertional mutants and constructing a platform of genetic transformation and functional genomics research in VerticiUium dahliae. [Method] There were 2628 T-DNA insertional mutants of Verticillium dahliae strain V991 and flanking sequences of 15 mutants were obtained, and the insertional sites of some mutants were analyzed by using Agrobacterium tumefaciens mediated transformation and high-efficiency TAIL-PCR, respectively. [Result] The mutant colony could be divided into three forms, which were hyphal type, sclerotium type and intermediate type. The sclerotium type colonies accounted for 7.3%. Culturing in flask, the sporulation peak of mutants was in the first 5-6 days after inoculation, and the sporulation ability of sclerotium mutants were generally higher than showing that of hyphal type mutants; VD991 could produce extracellular protease, amylase, cellulase and pectinase. Three mutants showing deleted protease secretion ability deleted, one mutant showing reduced amylase secretion ability and 6 mutants showing declined pectinase production were obtained; The virulence of the sclerotium type mutants was generally higher than that of the wild-type. Six mutants with reduced virulence were found from the virulence test of 26 mutants; The BLAST results showed that the flanking sequences of VD991 insertional mutants were 95%-100% identity to homologous sequences of Verticillium dahliae VDLs. 17, and 87%-94% identity to Verticillium alboatrum VaMs. 102. [ Conclusion ] Agrobacterium tumefaciens mediated T-DNA random insertion could be used to Verticillium dahliae mutagenesis, and the production of microsclerotia is related to virulence and sporulation. The American strain VDLs. 17 genome could be used as a reference sequence on genetic functional research of VD991.