生防链霉菌Men-myco-93-63遗传转化体系的建立和优化  

Optimized of the Conjugal Transfer System of Stremptomyces roseoflavus Men-myco-93-63

作　　者：沈凤英[1] 李亚宁[1] 刘力强 吴伟刚[3] 刘大群[1] 

机构地区：[1]河北农业大学植物保护学院,河北省植物病虫害生物防治工程技术研究中心,河北保定071001 [2]华北制药集团生物燃料研究所,石家庄050035 [3]河北北方学院,河北张家口075000

出　　处：《中国农学通报》2009年第13期197-201,共5页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金项目(No.30800734);河北省自然科学基金项目(No.C200900598);国家高技术研究与发展计划(863)(No.2006AA10A211)资助

摘　　要：玫瑰黄链霉菌(Streptomyces roseoflavus)Men-myco-93-63是分离自马铃薯疮痂病(S. scabies)自然衰退土壤中的一株拮抗菌。该菌株及其发酵液对棉花黄萎病菌(Verticillium dahliae)、瓜类白粉病菌(Sphaerotheca fuliginea Poll.)等多种重要的植物病原菌具有很强的抑制作用,有良好的生防应用潜力。为了对玫瑰黄链霉菌Men-myco-93-63中一些功能基因进行研究,得到高产抗生素的工程菌株,首先应对该菌的遗传转化条件进行优化。作者分别以基因整合型质粒pSET152和基因破坏型质粒pKC1139为出发质粒,ET12567(PUZ8002,pSET152/pKC1139)为供体,Men-myco-93-63孢子和菌丝体为受体,选取MS、PDA、TSB琼脂培养基、燕麦培养基为不同的培养基进行接合转移试验。结果表明MS培养基为接合转移的最适培养基,经验证,已成功地将质粒pSET152/pKC1139转入到了Men-myco-93-63中。以孢子为受体时,孢子预萌发条件为50℃热激10min,37℃温育2.5h,转化效率是10-7～10-6。以菌丝体为受体时,转化效率是10-7～10-6,但菌丝培养过程中容易出现污染。另外,抗生素的覆盖时间对接合转移效率的影响较明显,16～18h覆盖效果最好。Streptomyces roseoflavus Men-myco-93-63 was isolated from potato scab （S. scabies） decline soil and its fermentation can inhibit many phytopathogenic fungi and control related important plant diseases such as cotton verticillium wilt （Verticilliurn dahliae） and cumber powdery mildew （Sphaerothecafuliginea Poll.） In order to study a number of functional genes in Streptomyces roseoflavus Men-myco-93-63, and to achieve some engineering strain which producers of some important Conjugal Transfer System of this strain. In this article, The damage plasmid pKCl139 used as the starting plasmid, antibiotics. We must find the optimized of the genetic integrate plasmid pSET152 and genetic ET12567 （PUZS002, pSET152） and ET12567 （PUZ8002, pKCl139） used as the donor, Men-myco-93-63 spores and hyphae used as the receptor, respectively, the system for the conjugal transfer of pSET152 or pKCl139 from Escherichia coli in to Men-myco-93-63 has been developed. Different medium, such as MS, PDA, TSB agar, oats medium were selected for conjugal transfer, respectively. The results showed that MS medium is fit for conjugal transfer between Men-myco-93-63 and ET12567 （PUZ8002）, and it was confirmed that plasmid pSET152 and pKC 1139 had been successfully transferred to the Men-myco-93-63, respectively. When spores were used as the receptor, the pre-conditions of the germination of spores were 50℃ heat shock 10 min, 37℃ incubated 2.5 h in MS medium, and the conversion efficiency was 10-7 to 10-6. If the mycelium were used as the receptor, the conversion efficiency was 10-7 to l0-6, but the mycelium pollution can not be avoided. In addition, the coverage time on antibiotics effected the joint transfer efficiency significantly, 16 tol8 hours was best.

关 键 词：玫瑰黄链霉菌 接合转移 遗传转化 pKC1139 pSET152 

分 类 号：S182[农业科学—农业基础科学]