短小芽孢杆菌sRNA Bpsr112的鉴定及功能研究

Functional study on sRNA Bpsr112 of Bacillus pumilus

摘要:

为了给短小芽孢杆菌的改造提供新思路，提高碱性蛋白酶产量，实验室前期对短小芽孢杆菌进行了转录组测序，预测了短小芽孢杆菌的sRNA.本研究通过生物信息学方法和Northern杂交鉴定了一个新的sRNA Bpsr112.然后构建了Bpsr112的敲除型菌株和过表达菌株，利用相关菌株进行了生长曲线、盐胁迫和蛋白酶活等实验，再利用SDS-PAGE检测胞外蛋白酶AprE的表达水平.结果表明，在发酵至60 h和72 h时，与对照菌株相比，敲除型菌株酶活显著降低（P<0.01），过表达菌株酶活显著提高（P<0.01）；同时SDS-PAGE结果表明，AprE蛋白含量在敲除菌株中降低，在过表达菌株中提高，说明sRNA Bpsr112对短小芽孢杆菌蛋白酶活具有正调控.本研究鉴定了短小芽孢杆菌中的sRNA，并发现Bpsr112对蛋白酶活具有促进作用.

Abstract:

In order to provide new ideas for the transformation of Bacillus pumilus and increase the yield of alkaline protease, in our previous studies, transcriptome of Bacillus pumilus was analysed based on RNA-seq and sRNAs were predicted. In this study, a new sRNA Bpsr112 was identified by the bioinformatics analysis and northern blot.Then the knockout and overexpression strains of Bpsr112 were constructed, and the growth curve, salt stress and protease activity were compared among them. The results showed that, compared with the control strains at 60h and 72h, the protease activities of knockout strains were significantly decreased (P<0.01), while the protease activities of overexpression strains were significantly increased (P<0.01). At the same time, SDS-PAGE results showed that the content of AprE protein decreased in knockout strains and increased in overexpression strains. These results indicated that Bpsr112 may has positive regulation on the protease activity of Bacillus pumilus.In this study, sRNA in Bacillus pumilus was identified, and Bpsr112 was found to promote protease activity.