密旋链霉菌Act12遗传转化系统的建立与优化

Establishment and Optimization of Genetic Transfemation System for Streptomyces pactum Act12

密旋链霉菌(Streptomyces pactum)Act12是分离自黄土高原的1株多效放线菌,具有良好的生防应用价值,为了深入研究与利用,需建立该菌株的高效遗传转化系统。本试验以整合型质粒pSET152为出发质粒,大肠杆菌S17-1为供体,Act12的孢子为受体时,在改良2CMY培养基上进行接合转移,孢子预萌发条件为50℃热激10min,阿普霉素质量浓度为10.0mg/L,覆盖时间为18h,转化效率达到2.079×10^(-5)。以抗阿普霉素基因为目的基因,通过PCR验证,成功地将质粒pSET152转入到Act12中。同时,利用该转化系统,成功敲出Act12基因组中一可能的负调控转录因子spa0520,并获得了次级代谢图谱明显变化的缺失突变株Δspa0520。所构建的Act12遗传转化系统,为后续对其生防活性机理的研究以及深入开发利用奠定了基础。另外,通过培养基的改良发现,Act12在氮源含有NO-3时产孢丰富,为后续研究提供了思路。

Streptornyces pactum Actl2 was a multifunctional actinomycete strain isolated from an extreme environment in the Qinghai-Tibet Plateau, and it is valuble to apply in biological control. Devel- opment of a transformation system for Actl2 was needed to study the function of genes in Streptornyce pactum Actl2. In this paper, the genetic integration plasmid pSET152, E. coli S17-1, and Strepto- rnyce pactum Actl2 spores were used as the starting plasmid, the donor and the receptor, respective- ly. The results indicated that the pre-germination conditions of spores were heat shock at 50℃ for 10 minutes in the modified 2CMY medium. The mass concentration of apramycin was 10.0 rag/L, and the treatment time was 18 hours. The transformation efficiency was about 2. 079× 10-5. The apramycin gene was transferred and integrated successfully into Act12 genome as it was identified by PCR. The transcriptional repressors spa0520 in Actl2 was inactivated using the transformation system, then the mutant strainAspa0520 was obtained. The secondary metabolite profile inAspa0520 changed obvious- ly. The results showed that the genetic transformation system of Actl2 was constructed successfully, which it could lay the foundation for the subsequent research. In addition, we found that Act12 was rich in sporulation when the medium nitrogen source contained NO3- ,could provide guideline for further research.