利用电转化法构建烟草野火病菌的Tn5插入突变体  

Construction of Tn5 insertional mutants of Pseudomonas sringae pv. tabaci by electroporation

作　　者：陈红[1] 李江利[1] 焦伟[1] 蒋士君[1] 

机构地区：[1]河南农业大学植物保护学院,河南郑州450002

出　　处：《河南农业大学学报》2010年第3期322-325,共4页Journal of Henan Agricultural University

基　　金：河南省科技攻关重点项目(0623011900)

摘　　要：采用电转化法将含有kanr基因标记的EZ::Tn5转座子插入到烟草野火病菌(Pseudomonas syringae pv. tabaci)的基因组中.结果表明,在电压为2 400 V条件下,供试的烟草野火病菌菌株SMX 006及WT4的电转化效率分别为0.8×103,4.2×103cfu.μg-1DNA.转化电压下降至1 800 V时,WT4菌株的转化效率也随之下降至1.64×103cfu.μg-1DNA.对转化子进行kanr筛选以及特异性PCR鉴定,表明Tn 5可以稳定地插入到受体菌的基因组中.对烟草野火病菌SMX 006的Tn 5插入突变体进行了多次致病性、运动性和胞外蛋白酶活性测定,从中获得了2个致病力明显减弱的突变株(H028,H029)、1个运动能力明显减弱的突变株(H028)、1个运动能力丧失的突变株(H029)、1个产胞外蛋白酶能力明显减弱的突变株(H046),以及1个胞外蛋白酶缺失的突变株(H045).In this study,EZ：：Tn 5 carrying kanamycin resistance gene marker was inserted into genome of Pseudomonas syringae pv.tabaci by electroporation.The results showed that,under the condition of 2 400 volt,the transformation efficiency of two tested strains,SMX 006 and WT 4,was 0.8 × 103 and 4.2 × 103 cfu·μg-1 DNA,respectively.However,the trausformation efficiency of strain WT4 was decreased accordingly to 1.64 × 103 cfu·μg-1 DNA when the voltage for transformation was decreased to 1 800 volt.Screening by specific PCR-based identification and kanamycin resistance indicated that the EZ：：Tn5 could be inserted into transformants stably.By repeatable assay of pathogenicity,motility and extracellular protease secretion,some functional mutants were screened out from the EZ：：Tn5 insertional library： two mutants（H 028,H 029） with significantly weakened pathogenicity;mutant H 028 with reduced swimming motility,and mutant H 029 was motility-defective;mutant H 046 with significantly decreased extracellular protease secretion,and mutant H 045 could not secrete detectable extracellular protease.

关 键 词：烟草野火病菌 电转化 Tn5插入突变 功能突变体 

分 类 号：S432.42[农业科学—植物病理学]