生防菌Bs-916突变菌株M49芽胞形成和感受态形成能力  

Sporulation,competence development and biopesticide activity of a Bacillus subtilis mutant

作　　者：王晓宇[1] 罗楚平[1] 刘永锋[1] 刘邮洲[1] 聂亚锋[1] 陈志谊[1] 

机构地区：[1]江苏农业科学院植物保护研究所,南京210014

出　　处：《微生物学报》2009年第10期1295-1300,共6页Acta Microbiologica Sinica

基　　金：中国博士后科学基金(20080431108);江苏博士后基金(0801036B)~~

摘　　要：生防枯草芽胞杆菌Bs-916(Bacillus subtilis)在水稻纹枯病的防治上效果显著。应用离子注入突变对Bs-916进行了突变,获得了一系列的突变菌株。其中突变菌株M49,其表面活性素Surfactin分泌量比出发菌株Bs-916大大降低并导致其防效降低。【目的】为了确认影响该菌株防效降低的影响因子,对其表型和相关基因表达水平进行了研究。【方法】应用生孢培养基,通过芽胞形成能力评测方法比较该菌株和野生菌株Bs-916的芽胞形成能力;通过转化质粒的实验评测突变菌株M49和野生型Bs-916的感受态形成能力;应用RT-PCR(Semi-quantitative reverse transcriptase-polymerase chain-reaction)分析感受态形成重要的调控基因comS,影响芽胞形成的rapA、rapC基因的表达水平。【结果】发现二者的生长及生理有很明显的差异,突变菌株M49的芽胞形成能力大大降低了,和野生型Bs-916相比其芽胞数下降了约75%。而且其转化能力也大大减弱。在相同的转化条件下,M49转化效率下降了90%。RT-PCR结果显示M49突变菌株中未检测到comS、rapC基因的表达。通过基因互补获得了comA互补菌株,其抗真菌活性恢复到和野生型相似的水平。【结论】影响表面活性素surfactin合成的主要因子是两个,srfA操纵元启动子序列和comA基因。通过对二者的分析,发现comA基因发生了突变。结合M49的芽胞形成能力和感受态形成能力均受到了严重影响,且其芽胞形成相关基因rapC和感受态相关基因comS表达水平也大大下降的结果,提示我们导致M49表型发生变化的深层分子机理是否就是由于comA基因编码的ComA蛋白发生了点突变,从而导致ComA蛋白的生物活性发生改变,继而影响到相关调控过程。随后comA基因互补菌株又恢复了其抗真菌活性的证据支持了我们的上述推论。Bacillus subtilis Bs-916 has obvious effects against Rhizoctonia solani.We demonstrated that the mutant strain,M49 obtained by means of low energy ion implantation in strain Bs-916,which produces significantly lower levels of surfactin,had no obvious effects against R.solani.[Objective] In order to identify the influencing factors of surfactin-decrease mutant strain M49,its phenotype and related gene expression levels were studied.[Methods] Strains to be tested for sporulation were grown for 24 h in sporulation medium. Plasmid pDG1728（ 1μg/mL） was used for DNA transformation to test competence developmentof M49 and Bs-916 strains. RT-PCR （Semi-quantitative reverse transcriptase-polymerase chain-reaction） was used to determine the expression levels of selected genes involved in competence and sporulation in both the wildtype Bs-916 and mutant M49 strains, such as cornS, rapA, rapC gene. [ Results] Our data showed that mutant strain M49 confers a leaky competence phenotype, typified by a ten-fold reduction. This indicated a fact that DNA fragments are more easily transformed to wildtype strain than M49 mutant. The M49 strain also appeared to exhibit a sporulation-deficient phenotype, compared with the wild-type Bs-916, its spore＇s number declined by about 75%. RT-PCR results showed that both the cornS and rapC genes were expressed in the Bs-916 strain but not in the M49 strain. [Conclusions] Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of suffactin. Blast comparison of the sequences from Bs916 and M49 indicate that there is no difference in the srfA operon promoter, PsffA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 Coma protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild type Bs-916 and mutant M49 strains is significantly different. When

关 键 词：芽胞形成 感受态 表面活性素 基因突变 

分 类 号：Q93[生物学—微生物学] S476[农业科学—农业昆虫与害虫防治]