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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.
To determine the function of the cysDN operon and its effect on the nodulation of Rhizobia, the plasmid transposon pTnMod-RKm ’was randomly inserted into Sinorhizobium fredii 15142 by a three-parental conjugation and inserted into a library of mutants randomly generated, Screening MM medium with different sulfur sources resulted in a mutant that could not utilize sulfates but was able to utilize cysteine.After further cloning and sequencing analysis, it was found that the operon and the previously reported cysDN of Sinorhizobium sp. Strain BR816 92% similarity at the nucleotide level and 96% similarity at the amino acid level.The recombinant plasmid containing the cysD partial fragment and the cysN partial fragment was respectively constructed with the suicide plasmid pK18mob and introduced into the starting strain 15142 by the three parents, After homologous single exchange, the positive and negative pk18mob cysDF / 15142 and the pK18mob cysN / 15142 and cysNR / 15142 of cysDR / 15142 and cysN were respectively obtained by using homologous single exchange. Complement operon cysDN construct a complementary plasmid cysDN + pLAFRJ, the plasmid was introduced into the mutant strain by the three parents to obtain a complementary strain with different sulfur source liquid MM medium The results showed that the cysDN operon was indeed related to the pathway of sulfate assimilation. Plant experiments showed that the mutant strain delayed the onset of nodulation for 1 to 2 days than the original strain, and nitrogen fixation The activity of the enzyme was also slightly lower than that of the original strain. The competitive nodulation test showed that the mutant strains accounted for the poor tumor rate, but there was no difference in the average number of tumor, the average tumor weight and the average dry weight of plants.