刺糖多孢菌bldD基因过表达对多杀菌素合成及其孢子形成的影响  

Impact on Spinosad Biosynthesis and Spore Formation by Overexpression of bldD Gene in Saccharopolyspora spinosa

作　　者：蔡妹[1] 刘红雪[1] 杨琦[1] 孙运军[1] 胡胜标[1] 余子全[1] 黄伟涛[1] 丁学知[1] 夏立秋[1] 

机构地区：[1]湖南师范大学生命科学学院/微生物分子生物学国家重点实验室培育基地,长沙410081

出　　处：《中国生物防治学报》2015年第4期553-560,共8页Chinese Journal of Biological Control

基　　金：国家国际合作重点项目(2011DFA32610);国家"973"计划(2011CB7221301);国家"863"计划(2011AA10A203);湖南省协同创新中心项目(20134486)

摘　　要：bld D基因是链霉菌中的全局性调控基因,调节菌体的形态分化与次级代谢产物的合成。本研究采用重叠延伸PCR将bld D基因置于红霉素强启动子(Perm E)的控制下克隆至大肠杆菌-链霉菌穿梭载体p UC-spn上,构建重组载体p UC-spn-Perm E-bld D;通过接合转移将其导入刺糖多孢菌中,获得遗传性能稳定的重组菌株S.spinosa-Bld D。平板培养观察发现,在BHI和TSB平板上,重组菌株的孢子形成受到明显抑制。摇瓶发酵结果显示,重组菌株多杀菌素产量相比对照菌株提高了1.35倍。因此,bld D基因的过量表达在一定程度上抑制刺糖多孢菌的孢子形成,并有效促进多杀菌素的生物合成,为研究其他正调控基因的过量表达促进多杀菌素的生物合成奠定了重要基础。Previous report revealed that bldD was a global transcriptional regulator, participating in morphological development and secondary metabolism in Streptomyces. Here, we studied the effects of overexpression bldD gene on spinosad biosynthesis and spore formation in Saccharopolyspora spinosa. Cm-PermE gene segment and bldD gene were amplified, then spliced by overlap PCR. Thus, the open reading frame of bldD was placed under the control of the promoter for erythromycin resistance gene PermE. The cm-PermE-bldD frangment was cloned into Escherichia coli-Streptomyces shuttle vector pUC-spn by Red/ET homologous recombination, generating recombinant vector pUC-spn-PermE-bldD. This was introduced into S. spinosa from E. coli S17 through conjugal transfer, and integrated to the chromosome of S. spinosa via homologous recombination. Apramycin resistant colonies were picked up and identified by PCR. Positive transconjugants of which bldD gene was successfully integrated into chromosome of S. spinosa SP06081 were chosen for further study. On BHI and TSB agar, the sporulation of recombination strain S. spinosa-BldD was apparently inhibited. Flask fermentation demonstrated that the recombination strain overproduced 1.35-fold spinosad compared with the control strain. The recombination strain, S. spinosa-BldD, was genetically stable. These findings revealed that overexpression of bldD in S. spinosa had a negative effect on sporulation in some cases, and effectively promoted spinosad biosynthesis in S. spinosa, which provides a basis for improving spinosad production by overexpression of other positive regulatory genes.

关 键 词：刺糖多孢菌 bldD基因 过量表达 多杀菌素 

分 类 号：S476[农业科学—农业昆虫与害虫防治]