费氏中华根瘤菌HNO1 lrp基因的研究
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摘要
费氏中华根瘤菌HN01是一株快生型大豆根瘤菌，它的生长速度快、胞外多糖少，可用作共生固氮研究的模式菌株，也可作为商业化生产的菌剂。本工作运用分子生物学方法，通过原位杂交，从费氏中华根瘤菌HN01基因组文库中钓出含有lrp基因的阳性克隆，亚克隆并测定了lrp基因序列。生物信息学分析，HN01的lrp基因与已报道的苜蓿快生根瘤菌1021相比，在核苷酸水平上同源性有89％，在氨基酸水平上同源性有99％。
通过自杀质粒pK18mob同源单交换的方法构建了lrp基因的非极性突变体GXHNLTA和极性突变体GXHNLTB。通过PCR扩增，得到含有完整lrp基因ORF的片段lrpW，连结到pLAFR3上，获得互补质粒pGXHNL100。互补质粒为供体，通过三亲本接合导入突变株，获得互补株GXHNWA、GXHNWB。
在以脯氨酸、亮氨酸、丝氨酸等氨基酸为唯一碳、氮源的MM培养基中，突变体GXHNLTA、GXHNLTB的生长均滞后于出发菌株HN01。植株试验表明，突变体GXHNLTA、GXHNLB比出发菌株HN01开始结瘤时间提前1天，在结瘤效率、单株瘤数、瘤重、固氮酶活性方面均无显著差别。
Sinorhizobium fredii HN01 is a fast-growing Chinese isolate with fewerextracelluar polysaccher that has been adopted as a model organism for thestudy of nitrogen fixation and suited for commercial inoculation production. Inthis study, a positive clone containing lrp fragment of Sfredii HN01, wasisolated from DNA library by colony in Situ hybridization. A complete lrp genewas obtained through subcloning. Bioiformatics analysis showed that the lrpgene share 89% homology at nucleotide level and 99% homology at amino acidlevel with the reported lrp gene of Sinorhizobium meliloti 1021.
With the suicide plasmid pK18mob, polar mutant GXHNLTB andnon-polar mutant GXHNLTA inactivated in lrp gene have been constructedthrough homologous recombination caused by a single crossover event. Theintegrate lrp gene was amplified by PCR method and cloned into pLAFR3resulting in a complementati0n plasmid pGXHNL100. The complementationstrains GXHNWA and GXHNWB were constructed by transformingpGXHNL 100 into the host strains GXHNLTA and GXHNLTB, respectively.
Both non-polar and polar mutants of HN01 with lrp gene disrupted showed a little slower in growth with respect to their parent-type in MM mediumcontaining amino acid such as proline, leucine or serine as sole carbon andnitrogen source.
Plant tests revealed that the mutants GXHNLTA and GXHNLTB showed1 day ahead of the wild-type in initial nodulation time and its result did not showobvious effects of mutation of lrp gene on nodulation efficiency ability andnitrogen-fixation.
通过自杀质粒pK18mob同源单交换的方法构建了lrp基因的非极性突变体GXHNLTA和极性突变体GXHNLTB。通过PCR扩增，得到含有完整lrp基因ORF的片段lrpW，连结到pLAFR3上，获得互补质粒pGXHNL100。互补质粒为供体，通过三亲本接合导入突变株，获得互补株GXHNWA、GXHNWB。
在以脯氨酸、亮氨酸、丝氨酸等氨基酸为唯一碳、氮源的MM培养基中，突变体GXHNLTA、GXHNLTB的生长均滞后于出发菌株HN01。植株试验表明，突变体GXHNLTA、GXHNLB比出发菌株HN01开始结瘤时间提前1天，在结瘤效率、单株瘤数、瘤重、固氮酶活性方面均无显著差别。
Sinorhizobium fredii HN01 is a fast-growing Chinese isolate with fewerextracelluar polysaccher that has been adopted as a model organism for thestudy of nitrogen fixation and suited for commercial inoculation production. Inthis study, a positive clone containing lrp fragment of Sfredii HN01, wasisolated from DNA library by colony in Situ hybridization. A complete lrp genewas obtained through subcloning. Bioiformatics analysis showed that the lrpgene share 89% homology at nucleotide level and 99% homology at amino acidlevel with the reported lrp gene of Sinorhizobium meliloti 1021.
With the suicide plasmid pK18mob, polar mutant GXHNLTB andnon-polar mutant GXHNLTA inactivated in lrp gene have been constructedthrough homologous recombination caused by a single crossover event. Theintegrate lrp gene was amplified by PCR method and cloned into pLAFR3resulting in a complementati0n plasmid pGXHNL100. The complementationstrains GXHNWA and GXHNWB were constructed by transformingpGXHNL 100 into the host strains GXHNLTA and GXHNLTB, respectively.
Both non-polar and polar mutants of HN01 with lrp gene disrupted showed a little slower in growth with respect to their parent-type in MM mediumcontaining amino acid such as proline, leucine or serine as sole carbon andnitrogen source.
Plant tests revealed that the mutants GXHNLTA and GXHNLTB showed1 day ahead of the wild-type in initial nodulation time and its result did not showobvious effects of mutation of lrp gene on nodulation efficiency ability andnitrogen-fixation.
引文
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2．黄大防，林敏主编，《农业微生物基因工程》，科学出版社，2001
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