利用细菌表面展示技术构建镉耐受性的重组根瘤菌

Construction of Recombinant Rhizobial Strains with Enhanced Tolerance to Cadmium Using Bacterial Cell Surface Display*

通过细菌表面展示功能基因InaXN、猴金属硫蛋白α亚基四聚体MT4α和两种启动子PnifH及Plac元件,分别构建两个重组质粒pTNIM和pBLIM.进一步通过三亲本杂交,将重组质粒分别导入费氏中华根瘤菌HN01,分别构建获得诱导表达型重组菌HN01(pTNIM)和组成表达型重组菌HN01(pBLIM).在培养条件下比较测定重组菌和出发菌对镉的吸附能力和耐受性,结果表明,组成表达型重组菌HN01(pBLIM)在上述两个方面的能力得到明显增强,对镉的吸附富集能力提高了2倍.研究还发现重组根瘤菌也具有很好的静态吸附效果.盆栽实验结果表明,接种诱导表达型重组菌HN01(pTNIM)的大豆根瘤和根中Cd2+的富集量均明显高于野生菌株HN01.本实验为后续探索重组根瘤菌与宿主植物共生体系在Cd2+污染土壤修复中的应用前景奠定了基础.

This study aims to construct recombinant rhizobia strains with enhanced tolerance to cadmium by using bacterial cell surface displaying technique,and explore their application in bioremediation of Ca2＋ contamintated soil under symbiosis with the host leguminous plants.Two recombinant plasmids,pTNIM and pBLIM,were constructed based on the components of bacterial surface display functional gene InaXN,monkey metallothionein tetramer MT4α and two promoters of PnifH and Plac.By tri-parent matting technique,two recombinant plasmids were transferred into recipient Sinorhizobium fredii HN01.An inducible expression type of recombinant rhizobium HN01（pTNIM） and a constitutive expression type recombinant rhizobium HN01（pBLIM） were obtained,respectively.Under pure culture conditions,the adsorption capacity and tolerance ability of the recombinant and recipient strains to cadmium were comparatively examined,which showed the capacities for both aspects of HN01（pBLIM） were significantly improved,as such their cadmium-adsorption capacities were increased by over two folds.The results also indicated that the recombinant rhizobia demonstrated very good static-adsorption characteristics.Lastly,pot plant experiments showed that the amount of Cd2＋ absorbed in root nodules and roots of soybean plants inoculated with HN01 （pTNIM） was remarkably higher than that with wild-type strain HN01.