运动发酵单胞菌ZM4、CP4菌株基因工程选择标记的研究

摘要

尽管质粒和选择标记的使用作为基因工程最基本的一环而为人们所熟知,但对一些特殊菌种(菌株)或研究很少的菌种(菌株)的基因工程操作来说,质粒和选择标记可能仍然是一个并未完全解决的问题,因而需要不断提高认识、不断改进.运动发酵单胞菌Zymomonas mobilis具有突出的产醇性能,但其多种内源质粒和多种抗性的特点,增加了其基因工程操作时质粒和选择标记选用的难度.本研究在测定四个抗生素即Ap、Cm、Tc、Km对典型菌株ZM4、CP4的最低生长抑制浓度的基础上,初步确定了这两个菌株基因工程操作时的四个抗生素使用浓度依次分别为300、100、25、350μg/mL(ZM4)和500、100、25、250 μg/mL(CP4)；并进一步通过穿梭载体pZB21、宽宿主载体pBBR1MCS-2和整合载体pBR328-ldh R-cml - ldhL的转化,初步分析和证明了这些选择标记和在相应抗生素浓度下的效果:首先,对每一个选择标记基因来说,前述抗生素浓度是适于携带此选择标记基因的质粒的转化筛选和相应转化子培养的；其次,在前述抗生素浓度下,综合筛选平板阳性率和转化效率、培养物菌体形态异常程度等指标,四个选择标记基因中,以Cm和Tc抗性标记基因效果最好,Km抗性标记基因居中,Ap抗性标记基因最差.这些结果为ZM4、CP4基因工程遗传改造用抗性标记基因、质粒、抗生素的选择及转化系统的完善奠定了基础.%Using plasmid vector and selectable marker, as a fundamental and important step of genetic engineering, may still be a problem not being completely solved for some specific microorganism or strains. Zymomonas mobilis has excellent ethanol-producing capabilities, but its native plasmids and resistance to a variety of antibiotics make the selection of plasmid vector and selectable marker more difficult. In this study, the minimum inhibitory concentration of four antibiotic agents Ap, Cm, Tc and Km were tested for ZM4 and CP4 strains. Further the antibiotic concentrations for genetic engineering of two strains were primarily identified as 300, 100, 25 and 350 g/mL (for ZM4) and 500, 100, 25 and 250 g/mL (for CP4), respectively. The results to transform shuttle vector pZB21, broad-host-range vector pBER1MCS-2 into ZM4 and CP4, and integrating plasmid pER32&-ldhR-cml-ldhL into ZM4, showed the selected concentrations mentioned above were suitable for genetic engineering of ZM4 and CP4. On the other hand, the difference on the positive rate of colonies and transformation efficiencies of pZB21 in ZM4 or CP4 on different selective plates, the extent to be changed in the cell morphology for the transformant cell cultured in media with different antibiotics, all indicated that among four selectable marker genes, the best was Cmr- and Tcr-relating and the worst was Apr-relating one. Those results laid a preliminary foundation for selecting suitable resistance gene marker, plasmids and antibiotics, and establishing high-efficient genetic transformation system in Z. mobilis.