中文摘要：

为确定cysDN操纵子的功能及对根瘤菌结瘤的影响，通过三亲本接合，将质粒转座子pTnMod-RKm′随机插入费氏中华根瘤菌15142中，建成随机插入突变体库，随后通过含有不同硫源的MM培养基的筛选得到一株不能利用硫酸盐但能够利用半胱氨酸的突变体. 进一步克隆和测序分析后发现该操纵子与已报道的Sinorhizobium sp. strain BR816的cysDN在核苷酸水平上有92%的相似性，在氨基酸水平上有96%的相似性. 用自杀质粒pK18mob分别构建含有cysD部分片段和cysN部分片段的重组质粒，通过三亲本接合导入出发菌株15142中，经过同源单交换，分别获得cysD的pK18mob正反向插入突变株cysDF/15142 以及cysDR/15142和cysN的pK18mob正反向插入突变株cysNF/15142与cysNR/15142. 用广谱宿主质粒pLAFRJ载体连接完整操纵子cysDN构建互补质粒cysDN＋pLAFRJ，将该质粒通过三亲本接合导入突变株中，获得互补菌株. 用不同硫源的液体MM培养基培养，发现互补菌株能够补回突变菌株不能利用硫酸盐作为唯一硫源的缺陷，说明cysDN操纵子确实与硫酸盐同化途径有关；植株试验表明突变株比出发菌株推迟结瘤1-2 d，固氮酶活也比出发菌株稍低；竞争结瘤试验表明突变菌株占瘤率较差，但在平均瘤数、平均瘤重、平均植株干重上则无差异. 图3 表4 参10

英文摘要：

In order to know function of cysDN and its effect on plant nodulation, a random insertion mutant library of Sinorhizobium fredii 15142 was constructed by three parental hybridization with the plasmid pTnMod-RKm’ as transposon vector. A mutant strain which could not metabolize sulfate while could use cysteine as sulfur source, was selected by MM medium with different sulfur sources from the library. Molecular cloning and sequence analysis showed that this operon had 92% and 96% similarity with the cysDN gene sequence of Sinorhizobium sp. BR816 at nucleotide and amino acid level, respectively. With the suicide plasmid pK18mob, the polar mutant cysDF/15142, cysNF/15142 and non-polar mutant cysDR/15142, cysNR/15142 inactivated in cysD and cysN were constructed through homologous recombination. A recombinant plasmid pLAFRJ＋cysDN was constructed by using plasmid pLAFRJ ligated with cysDN, and transferred into above mutants to obtain complement strains. The results of growth tests found that the mutants had no growth on the MM medium with sulfate as sulfur source but the complement strains could grow normally, indicating that cysDN operon was related to sulfate assimilation. The plant tests indicated that the mutant strains delayed 1-2 d to nodulate than the wild strain, and nitrogenase activity and nodulation efficiency in mutants were slightly lower than those of the original strain, but there were no differences in the number and weight of nodules and the dry weight of plant. Fig 3, Tab 4, Ref 10