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

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trigger factor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。

Abstract:

[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 arms and constructed an integrative plasmid pKTF12 on the basis of plasmid pKSV7, a temperature sensitive plasmid. We also constructed a recombinant strain KCTF12 containing cry1Ac gene in its chromosome via the integrative plasmid pKTF12. [Results] Site-specific integration of cry1Ac into XBU001 chromosome did not affect its normal growth. The cry1Ac 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.