苏云金素合成基因簇中非核糖体肽合成酶基因thu2功能的初步分析

对苏云金素生物合成基因簇中编码非核糖体肽合成酶基因thu2进行基因缺失插入失活的研究。用温敏型质粒pHT304-TS构建基因thu2的插入缺失质粒pEMB1434,电转化苏云金芽胞杆菌菌株CT-43后,通过抗性筛选和PCR验证得到thu2基因同源双交换基因敲除突变株CT-43-22。HPLC(高效液相色谱,High Performance Liquid Chromatography)检测发现CT-43-22没有苏云金素特征吸收峰;用pHT304构建得到含有完整thu2基因的回补质粒pEMB1435,电转化CT-43-22后得到互补重组菌CT-43-22b,发现其恢复了苏云金素的产生。显微镜观察突变株和互补重组菌均能产生正常的晶体和芽胞。thu2的基因敲除和基因互补实验证明,thu2基因为CT-43苏云金素生物合成的必需基因,但对晶体和芽胞的形成没有影响。

Preliminary Analysis of the Nonribosomal Peptide Synthetase Gene thu2 in the Thuringiensin Biosynthesis Cluster

Thuringiensin is a small molecular weight nucleotide insecticidal toxin produced by some strains of Bacillus thuringiensis and secreted into extracellular supernatant. This work,on the base of revelation of the thuringiensin biosynthesis cluster in strain CT-43,preliminarily analyzed the function of gene thu2 in the cluster. Bioinformatics analysis indicated that thu2 was a nonribosomal peptide synthetase. We constructed plasmid pEMB1434 for thu2 knockout with temperature sensitive shuttle plasmid pHT304-TS and obtained the thu2 knockout mutant CT-43-22. HPLC detection found that the CT-43-22 could not produce thuringiensin. Then,we constructed plasmid pEMB1435 harboring intact thu2 and obtained the recombinant CT-43-22b after the pEMB1435 was electroporated. The CT-43-22b could restore the production of thuringiensin. Microscopic examination showed that the mutant and the recombinant could form natural spore and crystal. This research proved that the thu2 in the cluster is an integrant gene for thuringiensin biosynthesis,but unrelated to formations of spore and crystal.