外源基因在苏云金杆菌染色体上的定点整合及表达  

Site-specific integration of heterologous gene into Bacillus thuringiensis chromosome and its expression

作　　者：刘萍[1] 夏立秋[1,2] 胡胜标[1] 严礼[1] 丁学知[1] 张友明[1] 喻子牛[2] 

机构地区：[1]湖南师范大学生命科学学院,微生物分子生物学湖南省重点实验室,长沙410081 [2]华中农业大学,农业微生物学国家重点实验室,武汉430070

出　　处：《微生物学报》2008年第5期661-666,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30570050,30670052);国家“863计划”(2006AA02Z187,2006AA10A212);国家博士点基金项目(20060542006)~~

摘　　要：【目的】研究构建稳定表达外源基因、无抗性标记基因的苏云金杆菌(Bacillus thuringiensis,简称Bt)工程菌的方法。在构建Bt工程菌时,高拷贝外源质粒的转入导致Bt芽孢数量减少,芽孢形成期延滞,影响Bt菌株的杀虫活力。而且,外源质粒在Bt中的稳定性较差,外源基因容易丢失。将基因整合入染色体是一种构建遗传性状稳定、杀虫活力高的Bt工程菌的有效方法。【方法】本研究采用PCR技术,分两段扩增定位于Bt无晶体突变株XBU001染色体上的trigger factor基因片段作为同源臂,克隆入温度敏感型载体pKSV7,构建了定点整合载体pKTF12。并利用pKTF12质粒将cry1Ac基因定点整合入XBU001染色体上。【结果】利用载体pKTF12将cry1Ac定点插入triggerfactor位点,对宿主菌XBU001的正常生长没有影响。重组菌株KCTF12中的cry1Ac基因能够稳定遗传、表达并形成菱形晶体。与携带高拷贝外源质粒的Bt菌株HTX42相比较,KCTF12具有芽孢数量增多、芽孢形成期提前的优势。【结论】定点整合法是一种构建稳定表达外源基因、无抗性标记基因Bt工程菌的有效方法。[Objective] To efficiently construct resistance gene-free Bacillius thuringiensis engineered strain that can stably express heterologous gene. [Methods] We amplified the trigger factor gene located in chromosome of XBU001 strain as homologous alms and constructed an integrative plasmid pKTF12 on the basis of plasmid pKSV7, a temperature sensitive plasmid. We also constructed a recombinant strain KCTFI 2 containing crylAc gene in its chromosome via the integrative plasmid pKTFI2. [Results] Site-specific integration of crylAc into XBU001 chromosome did not affect its normal growth. The crylAc gene could stably express and form bipyramid crystals in KCTF12. When compared with HTX42 harboring a high-copy number plasmid, the recombinant strain KCTF12 has the merit of advanced sporulation and an increase in spore number. [Conclusion] The Site-specific integration proved to be a good approach to construct resistance gene-free Bacillius thuringiensis engineered strain that can stably express the heterologous gene.

关 键 词：苏云金芽孢杆菌 定点整合 同源重组 pKSV7 triggerfactor基因 CRY1AC基因 

分 类 号：S476.11[农业科学—农业昆虫与害虫防治]