与慢生大豆根瘤菌吸氢和固氮作用有关的基因克隆

CLONES OF THE DNA REGION INVOLVED IN BRADYRHIZOBIUM JAPONICUM HUP AND FIX FUNCTION

用Tn5插入导致的T-1(His^-Hup^-Fix^-)变株为受体,从慢生大豆根瘤菌USDA 110基因文库中钓取His^+结合子10株,经检测,它们都不同程度地恢复了Hup^+与Fix^+功能,从它们的DNA琼脂糖凝胶电泳图上,可见到都有一个分子量大小各异的pLAFRI::his质粒,其中5株接合子的重组质粒已转入E.coli HB101中,从它们的质粒电泳图型再显示其分子量各有差异,经Southern转移之后,分别用hup探针和nif探针进行DNA杂交分析,明确EC_r菌株的质粒能与hup和nif探针杂交,EC_6菌株的质粒能与hup探针杂交,其它三株虽能解除Hup^-Fix^-功能上的缺陷,但其质粒DNA序列上并无与nif或hup序列有同源性的成份。

Ten pL AFR1 cosmid clones carrying the His Hup Fix region of the Bradyrhizobium japonicum chromosome were isolated from a gene library of B. japonicum USDA 110 by complementation of a His^- Hup^- Fix^- mutant of B. Japonicum USDA110. Five of these recombinant plasmids were transferred into E. coli HB101 for further study. Using hup and nif probes, a plasmid containing a DNA region hybridyzing with hup and nif probes, another plasmid containing a DNA region hybridized only with hup probe were detected. Though the others did not hybridize with hup or nif probe, they did restore the Hup^+ Fix^+ function in His^- Hup^- Fix^- mutant.The results indicate that we have cloned at least three kinds of gene fragments coding for Hup and Fix activity.