转录因子cpcR同源基因cpcR-c1和cpcR-t的功能鉴定  

Functional characterization of cpcR-c1 and cpcR-t,homologous genes of transcription factor gene cpcR

作　　者：刘欢欢[1,2] 张睿彬 侯烁 彭琦 宋福平[1,2] LIU Huanhuan;ZHANG Ruibin;HOU Shuo;PENG Qi;SONG Fuping(College of Life Science,Northeast Agricultural University,Harbin 150030,Heilongjiang,China;State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150030 [2]中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京100193

出　　处：《微生物学报》2022年第5期1711-1721,共11页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31530095);中国博士后科学基金(2021M693463)。

摘　　要：苏云金芽胞杆菌(Bacillus thuringiensis,Bt)LM1212菌株与典型的Bt菌株表型不同,可分化形成芽胞、形成细胞和晶体产生细胞。在LM1212菌株中,转录因子CpcR不仅参与了细胞分化过程,而且能够激活晶体蛋白基因cry35-like的启动子(P_(35))。【目的】筛选cpcR同源基因,验证其生物学功能。【方法】本研究克隆了2个cpcR同源基因,来源于蜡样芽胞杆菌的cpcR-c1和来源于东洋芽胞杆菌的cpcR-t,将cpcR及其同源基因分别构建在pHT304-P_(35)-gfp、pHT304-P_(35)-lacZ报告载体上,获得的重组质粒转入无cpcR基因且无晶体蛋白基因的Bt HD73–菌株中。利用激光共聚焦显微镜观察重组菌HD–(cpcR-c1-P_(35)-gfp)和HD–(cpcR-t-P_(35)-gfp)的细胞表型并进行芽胞计数实验。测定HD–(cpcR-c1-P_(35)-lacZ)和HD–(cpcR-t-P_(35)-lacZ)菌株的β-半乳糖苷酶活性。【结果】与对照菌株相比,HD–(cpcR-c1-P_(35)-gfp)菌株和HD–(cpcR-t-P_(35)-gfp)菌株的芽胞细胞数分别降低了80.79%和90.14%,晶体产生细胞比例分别提高了7倍和9倍,并且gfp基因在这2个菌株中均能表达。β-半乳糖苷酶活性检测结果显示,启动子P_(35)在HD–(cpcR-c1-P_(35)-lacZ)菌株和HD–(cpcR-t-P_(35)-lacZ)菌株中有较高的转录活性。【结论】cpcR同源基因cpcR-c1、cpcR-t也能够影响细胞分化,激活P_(35)的转录,且cpcR-c1对P_(35)的激活能力大于cpcR。Compared with typical strains of Bacillus thuringiensis(Bt),LM1212 strain can differentiate into spore-formers and crystal-producers.In LM1212,the crystal-producing cell regulator(CpcR)not only participates in cell differentiation but also activates the promoter of crystal protein gene cry35-like(P_(35)).[Objective]We aimed to screen out the homologous genes of cpcR and verify their biological functions.[Methods]We cloned two cpcR homologous genes,cpcR-c1 from Bacillus cereus and cpcR-t from B.toyonensis.Then,we inserted cpcR and its homologous genes into pHT304-P_(35)-gfp and pHT304-P_(35)-lacZ vectors,respectively.The recombinant plasmids were transferred into Bt HD73−strain without cpcR and the crystal protein gene.We then observed the cell phenotypes of recombinant strains HD−(cpcR-c1-P_(35)-gfp)and HD−(cpcR-t-P_(35)-gfp)by using a laser confocal microscope and quantified the sporulation efficiency.Theβ-galactosidase activities of HD−(cpcR-c1-P_(35)-lacZ)and HD−(cpcR-t-P_(35)-lacZ)strains were determined.[Results]Compared with the control strain,strain HD−(cpcR-c1-P_(35)-gfp)and HD−(cpcR-t-P_(35)-gfp)showed the number of spores decreasing by 80.79%and 90.14%and the percentage of crystal-producers increasing by 7 and 9 times,respectively.Gene gfp was expressed in these two strains.Theβ-galactosidase activity assay demonstrated that promoter P_(35) had high transcriptional activity in strain HD−(cpcR-c1-P_(35)-lacZ)and HD−(cpcR-t-P_(35)-lacZ).[Conclusion]The homologous genes of cpcR,cpcR-c1 and cpcR-t can regulate cell differentiation and activate the transcription of P_(35),and cpcR-c1 had better performance in activating P_(35) transcription than cpcR.

关 键 词：苏云金芽胞杆菌 cpcR-c1 cpcR-t 细胞分化 

分 类 号：Q78[生物学—分子生物学]