利用荧光指示分子IeGFP比较vip3A和cry1Ia基因启动子活性  

Comparison of the Promoter Activity of vip3A and cry1Ia using IeGFP as a Fluorescent Reporter

作　　者：高建华[1] 欧阳春平 钱红梅 刘小琼 郭俊佩 赵雄伟 王兴春[1] 韩渊怀[3] 李旭凯 侯思宇[3] GAO Jian-Hua;OUYANG Chun-Ping;QIAN Hong-Mei;LIU Xiao-Qiong;GUO Jun-Pei;ZHAO Xiong-Wei;WANG Xing-Chun;HAN Yuan-Huai;LI Xu-Kai;HOU Si-Yu(College of Life Sciences,Shanxi Agricultural University,Taigu 030801,Shanxi,China;State Key Laboratory of Natural Medicines,School of Traditional Chinese Pharmacy,China Pharmaceutical University,Nanjing 211198,China;College of Agriculture,Shanxi Agricultural University,Taigu 030801,Shanxi,China)

机构地区：[1]山西农业大学生命科学学院,山西太谷030801 [2]中国药科大学中药学院天然药物国家重点实验室,南京211198 [3]山西农业大学农学院,山西太谷030801

出　　处：《中国生物化学与分子生物学报》2021年第5期617-626,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.31601690);山西省应用基础研究面上青年基金项目(No.201901D211362);山西农业大学科技创新基金项目(No.2017YJ27)资助。

摘　　要：苏云金芽孢杆菌(Bt)的Vip3A和Cry1Ia蛋白质序列无同源性,但具有其他相似的特征,例如在孢子形成早期合成,然后跨膜转运至胞外。本研究以新型融合荧光蛋白IeGFP为报告分子,比较vip3A(Pv)和cry1Ia基因(Pi)启动子的调控模式和活性。结果表明,在大肠杆菌中,两者均为组成型启动子。通过荧光信号强度的比较发现,在大肠杆菌中,Pi活性显著强于乳糖操纵子调节基因启动子(PlacI,P<0.01),但比PlacI增强型突变体(PlacIq,P<0.01)和cry1Ac基因启动子(Pac,P<0.01)弱。在3个Bt启动子中,Pv活性最弱,接菌12 h后,与PlacI接近(P>0.05)。在Bt菌株中,Pv对IeGFP的表达调控与之前报道的Pi相似。启动子的Shine-Dalgarno(SD)序列与目的基因起始密码子(AUG)之间的间隔区域在调节细菌的蛋白质翻译效率方面发挥重要作用。本研究中构建的大部分Pv与Iegfp基因的间隔区域突变,均导致相应大肠杆菌和Bt细胞的荧光强度显著降低(P<0.05),说明融合荧光蛋白IeGFP具有较好的灵敏度。该研究不仅确定了2种启动子在Bt和大肠杆菌中的活性,而且验证了IeGFP指示弱启动子活性的可行性。In spite of no homology in sequences,Vip3A and Cry1Ia toxins of Bacillus thuringiensis (Bt)share common characteristics,such as translocation across cell membranes after synthesis at the early stage of sporulation.The aim of the present study was to compare the regulation patterns and activities of the promoters of vip3A (Pv) and cry1Ia genes (Pi) using the novel fusion fluorescent protein IeGFP as the reporter molecule.The result showed that in E.coli their constitutive regulations were identified for the first time.By comparing the fluorescent intensities,we found that Pishowed significantly stronger activity than the lactose repressor gene promoter (PlacI,P<0.01) at 12 hours after inoculation but weaker activity than the‘up’mutant of the PlacI(PqlacI,P<0.01) and cry1Ac gene promoters (Pac,P<0.01) in E.coli.Among the three Bt promoters,Pvwas the weakest and showed similar levels to PlacI(P>0.05) at 12hours after inoculation.In Bt,Pvhad the similar patterns in regulating the expression of IeGFP to Pias reported previously.The spacer region between the Shine-Dalgarno (SD) sequence of a promoter and the initiation codon (AUG) of target genes play an important role in regulating translation efficiency in bacteria.Most of alterations in this region of Pvin this study resulted in significant decreases (P<0.05)in the fluorescent intensity of both E.coli and Bt cells expressing the IeGFP protein,suggesting the sensitivity of the fusion fluorescent protein.The study not only determined the transcriptional activities of two Bt promoters that can be used in both Bt and E.coli strains,but also identified the suitability of IeGFP as an activity indicator of the promoter with weaker activity.

关 键 词：苏云金芽孢杆菌 vip3A cry1Ia 启动子活性 荧光强度 

分 类 号：S182[农业科学—农业基础科学]