【作者】 龙章德；

【导师】 武波；

【作者基本信息】 广西大学 ， 微生物学， 2002， 硕士

【摘要】 本研究首先利用转座子Tn5gusA5对重组质粒pGXN201进行诱变，获得3.4kbEcoRI片段插入了Tn5gusA5的突变质粒pGXN215、pGXN216、pGXN217。对这些突变质粒进行亚克隆及测序分析并与基因库中已报道的慢生型大豆根瘤菌的DNA序列作比较，发现突变质粒pGXN217中Tn5gusA5恰好插入在一个未知功能的开放阅读框架内。将pGXN201的3.4kb EcoRI片段与M13作连接，获得亚克隆pGXN201C，对pGXN201C进行测序分析，发现与基因库中已报道的慢生型大豆根瘤菌的DNA序列有95％的同源性。随后，利用突变质粒pGXN217诱变慢生型大豆根瘤菌菌株GX201，获得了GX201的突变体菌株GX217；将pGXN201的3.4kbEcoRI片段与pLARF3连接获得亚克隆pGXN201cl，然后将pGXN201cl导入突变体菌株GX217，构建了GX217的功能互补菌株rGX217。 通过对野生型菌株GX201，突变体菌株GX217及其功能互补菌株rGX217进行植株试验，发现突变体菌株GX217在结瘤时间方面较野生型菌株GX201慢一天，其结瘤效率及竞争结瘤能力均比GX201明显降低。我们认为在突变体菌株GX217中Tn5gusA5所插入的开放阅读框架是一个新的与竞争结瘤有关的基因。更多还原

【Abstract】 In this study, a recombinant plasmid pGXN201 was first mutated by using of transposon Tn5gusA5 mutagenesis, and three mutant plasmids , pGXN215, pGXN216 and pGXN217 were achieved. The 3.4kb EcoRI DNA fragment of the pGXN201 and the mutant EcoRI DNA fragments of the three mutant plasmids were subcloned into the EcoRI sites of the clone vectors Ml3 and the pGEM3Z-f(+) respectively.Through DNA sequencing and DNA sequence analysis by comparing with the Bradyrhizobium Japonicum DNA sequences reported, we found that there are four intact Open Reading Frames in the 3.4kb fragment and it was highly homologous to the Bradyrhizobium Japonicum DNA sequence reported, and the Tn5gusA5 containing in the pGXN217 was inserted in one of the four Open Reading Frames .This Open Reading Frame located on about 5.5kb upstream of the nfeC gene, which includes 207bp nucleotides, coding 68 putative amino acids and this is completely identical to that of the Bradyrhizobium Japonicum DNA sequence reported.pGXN217 was transferred into Bradyrhizobium Japonicum strain GX201 by triparental mating using the helper plasmid pRK2073. Marker exchange was achieved by selection on YMA containing Sm, Km ,Gm, Spc et al four kinds of antibiotics. Mutants were confirmed by southern blotting and hybridization with the wild-type region and the Tn5 fragment respectively. The mutant strain GX217 was achieved. The 3.4kb EcoRI fragment of the plasmid pGXN201 was firstly subcloned into the EcoRI site of the plARF3, then the recombinant plasmid was introduced into the mutant strain GX217 by triparental mating using the helper pRK2073 and the mutant strain rGX217 was achieved.Plant assay was performed with the wild-type strain GX201,the mutant strain GX217 and the function recovered strain rGX217.The results indicate that the mutant strain GX217 showed a day delay in nodulation time and its abilities of nodule formation efficiency and competitive nodulation were apparently decreased compared with the parental strain GX201 respectively. We conclude that the gene we studied is a new loci required for efficient nitrogen fixation and for competitive nodulation of soybeans by Bradyrhizobium Japonicum strain GX201.更多还原