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

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(P₃₅)。【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-P₃₅-gfp、pHT304-P₃₅-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-P₃₅-gfp)和HD^–(cpcR-t-P₃₅-gfp)的细胞表型并进行芽胞计数实验。测定HD^–(cpcR-c1-P₃₅-lacZ)和HD^–(cpcR-t-P₃₅-lacZ)菌株的β-半乳糖苷酶活性。【结果】与对照菌株相比，HD^–(cpcR-c1-P₃₅-gfp)菌株和HD^–(cpcR-t-P₃₅-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P₃₅在HD^–(cpcR-c1-P₃₅-lacZ)菌株和HD^–(cpcR-t-P₃₅-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P₃₅的转录，且cpcR-c1对P₃₅的激活能力大于cpcR。

Abstract:

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 genecry35-like (P₃₅). [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-P₃₅-gfp and pHT304-P₃₅-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-P₃₅-gfp) and HD⁻(cpcR-t-P₃₅-gfp) by using a laser confocal microscope and quantified the sporulation efficiency. The β-galactosidase activities of HD⁻(cpcR-c1-P₃₅-lacZ) and HD⁻(cpcR-t-P₃₅-lacZ) strains were determined. [Results] Compared with the control strain, strain HD⁻(cpcR-c1-P₃₅-gfp) and HD⁻(cpcR-t-P₃₅-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. The β-galactosidase activity assay demonstrated that promoter P₃₅ had high transcriptional activity in strain HD⁻(cpcR-c1-P₃₅-lacZ) and HD⁻(cpcR-t-P₃₅-lacZ). [Conclusion] The homologous genes of cpcR, cpcR-c1 and cpcR-t can regulate cell differentiation and activate the transcription of P₃₅, and cpcR-c1 had better performance in activating P₃₅ transcription than cpcR.