苏云金芽胞杆菌cry8Ea启动子区orf1片段缺失增强启动子活性
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摘要
本文分别构建了cry8E基因上游的启动子(Porf18E)和其上游缺失orf1基因的启动子(PΔorf18E)融合lacZ基因的表达载体,通过β-半乳糖苷酶活性的分析,发现PΔorf18E的转录活性高于Porf18E。分别用PΔorf18E和Porf18E指导cry1Ac基因的表达,通过光学显微镜观察,发现两个启动子指导表达的Cry1Ac蛋白均可形成双锥形晶体;通过总蛋白定量分析发现,缺失orf1基因的启动子(PΔorf18E)指导的Cry1Ac蛋白表达量高于Porf18E启动子指导的Cry1Ac蛋白表达量;生物活性测定表明:PΔorf18E指导的Cry1Ac晶体蛋白对小菜蛾Plutella xylostella具有杀虫活性,高于Porf18E指导的Cry1Ac晶体蛋白对小菜蛾的杀虫活性。本文获得的强活性的启动子PΔorf18E是目前已报道的转录活性最高的cry基因启动子,该启动子为Cry蛋白的表达和遗传工程菌株构建提供了重要元件。
The cry8E promoter(Porf18 E) and the promoter(PΔorf18 E) with the deletion of orf1 fragment, located upstream of cry8E, were fused with lacZ gene. The transcriptional activity of PΔorf18 E was higher than that of Porf18 E. The expression of Cry1 Ac was directed by PΔorf18 E and Porf18 E, and the strains HD~-(Porf18 E-1 Ac) and HD~-(PΔorf18 E-1 Ac) were obtained. Both strains could produce the typical bipyramidal crystals, while HD~-(PΔorf18 E-1 Ac) strain produced more Cry1 Ac, which was more toxic to Plutella xylostella than HD~-(Porf18 E-1 Ac). PΔorf18 E has the highest transcriptional activity in cry gene promoters. It provides an important element for construction of engineered Bt strains.
The cry8E promoter(Porf18 E) and the promoter(PΔorf18 E) with the deletion of orf1 fragment, located upstream of cry8E, were fused with lacZ gene. The transcriptional activity of PΔorf18 E was higher than that of Porf18 E. The expression of Cry1 Ac was directed by PΔorf18 E and Porf18 E, and the strains HD~-(Porf18 E-1 Ac) and HD~-(PΔorf18 E-1 Ac) were obtained. Both strains could produce the typical bipyramidal crystals, while HD~-(PΔorf18 E-1 Ac) strain produced more Cry1 Ac, which was more toxic to Plutella xylostella than HD~-(Porf18 E-1 Ac). PΔorf18 E has the highest transcriptional activity in cry gene promoters. It provides an important element for construction of engineered Bt strains.
引文
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[2] 李朝睿,杜立新,彭琦,等.苏云金芽胞杆菌高效表达载体的构建[J].微生物学通报,2013,40(2):350-361.
[3] 郑庆云,王冠男,张喆,等.芽胞外壁基质组成蛋白的编码基因启动子PexsY指导的cry1Ac基因表达[J].微生物学报,2014,10:005.
[4] ZHANG Xin, GAO Tantan, PENG Qi, et al. A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis[J]. Applied Microbiology and Biotechnology, 2018,102(8):3687-3699.
[5] DU Lixin, QIU Lili, PENG Qi, et al. Identification of the promoter in the intergenic region between orf1 and cry8Ea1 controlled by sigma H factor [J]. Applied and Environmental Microbiology, 2012, 78(12): 4164-4168.
[6] ZHOU Changmei, ZHENG Qinyun, PENG Qi, et al. Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant [J]. Applied Microbiology and Biotechnology, 2014, 98(18): 7901-7909.
[7] SCHAEFFER P, MILLET J, AUBERT J P. Catabolic repression of bacterial sporulation [J]. Proceedings of the National Academy of Sciences, 1965, 54(3): 704-711.
[8] SHU Changlong, YU Hong, WANG Rongyan, et al. Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185 [J].Current Microbiology,2009,58(4):389-392.
[9] AGAISSE H, LERECLUS D. Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis[J]. Molecular Microbiology, 1994, 13(1): 97-107.
[10] LIU Guiming, SONG Lai, SHU Changlong, et al. Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73 [J]. Genome Announcements,2013,1(2):e00080-13.
[11] PERCHAT S, DUBOIS T, ZOUHIR S, et al. A cell-cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group [J]. Molecular Microbiology, 2011, 82(3): 619-633.
[12] 王新梅,杜立新,彭琦,等.四种cry基因启动子在spoIIID基因突变株中的活性比较[J].微生物学报,2012,52(9):1075-1084.
[13] BARBOZA-CORONA J E, PARK H W, BIDESHI D K, et al. The 60-kilodalton protein encoded by orf2 in the cry19A operon of Bacillus thuringiensis subsp. jegathesan functions like a C-terminal crystallization domain[J]. Applied and Environmental Microbiology, 2012, 78(6): 2005-2012.
[14] DERVYN E, PONCET S, KLIER A, et al. Transcriptional regulation of the cryIVD gene operon from Bacillus thuringiensis subsp. israelensis[J]. Journal of Bacteriology, 1995, 177(9): 2283-2291.
[15] WIDNER W R, WHITELEY H R. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities [J]. Journal of Bacteriology, 1989, 171(2): 965-974.
[16] DENG Chao, PENG Qi, SONG Fuping, et al. Regulation of cry gene expression in Bacillus thuringiensis[J]. Toxins, 2014, 6(7): 2194-2209.
[17] STAPLES N, ELLAR D, CRICKMORE N. Cellular localization and characterization of the Bacillus thuringiensis Orf2 crystallization factor [J]. Current Microbiology, 2001, 42(6): 388-392.
[18] YU Ziquan, BAI Peisheng, YE Weixing, et al. A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis [J]. Journal of Microbiology and Biotechnology,2008,18(6):1033-1039.
[2] 李朝睿,杜立新,彭琦,等.苏云金芽胞杆菌高效表达载体的构建[J].微生物学通报,2013,40(2):350-361.
[3] 郑庆云,王冠男,张喆,等.芽胞外壁基质组成蛋白的编码基因启动子PexsY指导的cry1Ac基因表达[J].微生物学报,2014,10:005.
[4] ZHANG Xin, GAO Tantan, PENG Qi, et al. A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis[J]. Applied Microbiology and Biotechnology, 2018,102(8):3687-3699.
[5] DU Lixin, QIU Lili, PENG Qi, et al. Identification of the promoter in the intergenic region between orf1 and cry8Ea1 controlled by sigma H factor [J]. Applied and Environmental Microbiology, 2012, 78(12): 4164-4168.
[6] ZHOU Changmei, ZHENG Qinyun, PENG Qi, et al. Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant [J]. Applied Microbiology and Biotechnology, 2014, 98(18): 7901-7909.
[7] SCHAEFFER P, MILLET J, AUBERT J P. Catabolic repression of bacterial sporulation [J]. Proceedings of the National Academy of Sciences, 1965, 54(3): 704-711.
[8] SHU Changlong, YU Hong, WANG Rongyan, et al. Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185 [J].Current Microbiology,2009,58(4):389-392.
[9] AGAISSE H, LERECLUS D. Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis[J]. Molecular Microbiology, 1994, 13(1): 97-107.
[10] LIU Guiming, SONG Lai, SHU Changlong, et al. Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73 [J]. Genome Announcements,2013,1(2):e00080-13.
[11] PERCHAT S, DUBOIS T, ZOUHIR S, et al. A cell-cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group [J]. Molecular Microbiology, 2011, 82(3): 619-633.
[12] 王新梅,杜立新,彭琦,等.四种cry基因启动子在spoIIID基因突变株中的活性比较[J].微生物学报,2012,52(9):1075-1084.
[13] BARBOZA-CORONA J E, PARK H W, BIDESHI D K, et al. The 60-kilodalton protein encoded by orf2 in the cry19A operon of Bacillus thuringiensis subsp. jegathesan functions like a C-terminal crystallization domain[J]. Applied and Environmental Microbiology, 2012, 78(6): 2005-2012.
[14] DERVYN E, PONCET S, KLIER A, et al. Transcriptional regulation of the cryIVD gene operon from Bacillus thuringiensis subsp. israelensis[J]. Journal of Bacteriology, 1995, 177(9): 2283-2291.
[15] WIDNER W R, WHITELEY H R. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities [J]. Journal of Bacteriology, 1989, 171(2): 965-974.
[16] DENG Chao, PENG Qi, SONG Fuping, et al. Regulation of cry gene expression in Bacillus thuringiensis[J]. Toxins, 2014, 6(7): 2194-2209.
[17] STAPLES N, ELLAR D, CRICKMORE N. Cellular localization and characterization of the Bacillus thuringiensis Orf2 crystallization factor [J]. Current Microbiology, 2001, 42(6): 388-392.
[18] YU Ziquan, BAI Peisheng, YE Weixing, et al. A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis [J]. Journal of Microbiology and Biotechnology,2008,18(6):1033-1039.