论文部分内容阅读
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 (Bacillus subtilis) in the control of rice sheath blight significant effect. Bs-916 was mutated using ion-implantation mutation and a series of mutant strains were obtained. The mutant strain M49, Surfactin Surfactin secretion than the original strain Bs-916 greatly reduced and lead to its reduced control. 【Objective】 The objective of this study was to investigate the phenotypic and related gene expression levels in order to identify the influencing factors that affect the control efficacy of this strain. 【Method】 The spore-forming medium was used to compare the sporulation ability of this strain and wild strain Bs-916 by the method of sporulation assay. The competent cell of M49 and wild-type Bs-916 were evaluated by plasmid transformation. Semi-quantitative reverse transcriptase-polymerase chain-reaction (RT-PCR) was used to analyze the competent cells to form an important regulatory gene, comS, which affected the expression of rapA and rapC genes. 【Result】 The results showed that the growth and physiology of the two strains were obviously different. The spore forming ability of mutant strain M49 was greatly reduced. Compared with wild type Bs-916, its spore number decreased about 75%. And its conversion ability is greatly reduced. Under the same conversion conditions, M49 conversion efficiency decreased by 90%. The results of RT-PCR showed that the expression of comS and rapC gene was not detected in M49 mutant strains. Complementary strains of comA were obtained by gene complementation and their antifungal activity returned to similar levels as the wild type. 【Conclusion】 The two main factors that affect the synthesis of surfactant surfactin are srfA promoter promoter sequence and comA gene. Through the analysis of the two, we found that there was a mutation in the comA gene. The ability of spleen formation and competence formation in combination with M49 were severely impaired, and the expression of rapC and comS, a gene related to spore formation, was also greatly reduced, suggesting that we have a deep molecular mechanism of M49 phenotypic changes Whether it is due to comA gene encoded ComA protein point mutation, resulting in changes in the biological activity of ComA protein, which in turn affect the relevant regulatory processes. Subsequent evidence that the comA gene complementation strain has regained its antifungal activity supports our above reasoning.