生防链霉菌gCLA4遗传转化体系的建立与优化

Establishment and optimization of genetic transformation system for bio-control Streptomyces lavendularectus gCLA4

【目的】建立生防菌淡紫灰链霉菌(Streptomyces lavendularectus)gCLA4的遗传转化体系,为定向改造基因、提高基因表达水平及其生防分子机制研究奠定基础。【方法】以具有安普霉素(Apramycin)抗性基因标记的整合型质粒pSET152为出发质粒,Escherichia coli ET12567(pUZ8002,pSET152)为供体,淡紫灰链霉菌gCLA4为受体进行接合转移试验,并对影响接合效率的培养基、热激温度、接合转移时间和筛选转化子培养基等因素进行优化。【结果】成功地将质粒pSET152转入到了生防链霉菌gCLA4中,明确了可用于筛选链霉菌gCLA4转化子的抗生素为安普霉素或四环素;链霉菌gCLA4孢子50℃热激时转化效率较高,接合效率在16,18,20h下没有明显差异,MS培养基作为接合转移培养基时转化效率最高,接合产物涂布到高氏一号抗性培养基上进行阳性转化子筛选的效果最好。【结论】建立了生防链霉菌gCLA4的遗传转化优化体系。

【Objective】The genetic transformation system of bio-control Streptomyces lavendularectus gCLA4 was established for modifying gene and raising the level of gene expression and laying foundation for research of its bio-control mechanisms.【Method】Intergeneric genetic transfer system was based upon integrative plasmid pSET152 with apramycin resistance gene from donor Escherichia coli ET12567/pUZ8002 to S.lavendularectus gCLA4.Various influencing factors including culture medium,heat temperature,time of conjugation and the medium for screening were optimized.【Result】The plasmid was transferred into S.lavendularectus gCLA4 and the best conditions of conjugation were determined.Apramycin and tetracycline could be used to screen the transformation of gCLA4.When the gCLA4 spores were heatshocked at 50 ℃,the conjugation efficiency was the highest.There was no difference in conjugation among different times 16,18,and 20 h.MS medium was the preferred medium for conjugation and exconjuantscoating to the GAUZE＇s medium No.1 with antibiotic was the best.【Conclusion】The optimal genetic transformation system of bio-control S.lavendularectus gCLA4 was established.