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

• Published in: Henan nongye daxue xuebao Vol. 44; no. 3
• Main Authors: Chen Hong, Henan Agricultural University,Zhengzhou(China), College of Plant Protection, Li Jiangli, Henan Agricultural University,Zhengzhou(China), College of Plant Protection, Jiao Wei, Henan Agricultural University,Zhengzhou(China), College of Plant Protection
• Format: Journal Article
• Language: Chinese
• Published: 01.05.2010
• Subjects: ELECTROPORACION; ELECTROPORATION; http://www.fao.org/aos/agrovoc#c_26650; http://www.fao.org/aos/agrovoc#c_33983; http://www.fao.org/aos/agrovoc#c_5013; MUTANT; MUTANTES; MUTANTS; PSEUDOMONAS SYRINGAE
• ISSN: 1000-2340

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）.[著者文摘] 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