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从快生型大豆根瘤菌费氏中华根瘤菌HN01的基因组文库中克隆到一个putA(脯氨酸脱氢酶)基因,该基因与已报道的苜蓿中华根瘤菌1021中putA基因在核苷酸和氨基酸水平上分别有82%和86%相似性.利用自杀性质粒pK18mob构建含有putA基因部分片段的重组质粒,通过三亲本结合导入出发菌株HN01中,获得正向插入的极性突变体GXHNPA和反向插入非极性突变体GXHNPB.将putA基因完整的ORF连接到广宿主载体pLAFRJ上,获得用于互补的质粒pGXHN37,通过三亲接合,将pLAFRJ导入GXHNPA和GXHNPB获得互补菌株GXHNPHA和GXHNPHB.对出发菌株、突变菌株、互补菌株进一步的研究发现,以脯氨酸为唯一C、N源的MM培养基中培养时,突变体均不能生长,互补菌株和野生型HN01没有差异,而在完全培养基YMB中培养时,突变菌株生长不受影响.植株实验发现,突变体均能有效结瘤,但是固氮酶活与出发菌株相比有所下降,结瘤时间延迟1d,竞争结瘤能力下降,而互补菌株与野生型HN01没有明显的差异.
A putA (proline dehydrogenase) gene was cloned from the genomic library of fast-growing soybean Rhizobium fredii Sinorhizobium HN01. The putA (proline dehydrogenase) gene was cloned from the putA gene of Sinorhizobium meliloti 1021 Amino acid levels were 82% and 86% respectively.Using the suicide plasmid pK18mob to construct a recombinant plasmid containing part of the putA gene, the recombinant plasmid was introduced into the parental strain HN01 by a three-parental union to obtain the positive insert polar mutant GXHNPA and the reverse The non-polar mutant GXHNPB was inserted into the vector pLAFRJ of the putA gene to obtain the plasmid pGXHN37 for complementation, and the complemented strains GXHNPHA and GXHNPHB were obtained by introducing pLAFRJ into GXHNPA and GXHNPB through paternal mating Further study of starting strain, mutant strain and complementation strain showed that mutants could not grow when grown in MM medium containing proline as sole C and N source. There was no difference between the complementary strain and wild type HN01, The growth of mutant strains was not affected when cultured in base YMB.The plant experiments showed that all the mutants could effectively nodulate, but the activity of nitrogenase activity decreased compared with that of the original strain, and the nodulation time Chi 1d, competitive nodulation ability to decline, while the complementary wild-type strain HN01 no significant difference.