野油菜黄单胞菌gum基因簇DNA片段上基因间隔区中的ORF及其功能分析
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摘要
利用RT—PCR的方法，对野油菜黄单胞菌致病变种(Xanthomonas campestris
pv.campestris)8004(Xcc8004)及其Tn5gusA5转座子插入gum基因的突变体的gum基因操纵子的转录进行了比较分析。结果表明Tn5gusA5的插入具有很强的完全极性效应，并进一步证实gumK，gumL和gumM基因构成了gum基因操纵子中第二个转录本。
通过TAIL—PCR和测序方法对转座子Tn5gusA5插入突变体TGL243，TGL14，TGL233和TGL40中转座子Tn5gusA5在Xcc8004基因组中的位置进行了定位。并对这四株突变体的一些生物学特性和流变学特性进行了测定和分析，结果表明突变体TGL243色素产量约为Xcc8004色素产量的34％，TGL40约为Xcc8004的130％。TGL233胞外多糖的组分和粘度与Xcc8004不同，其胞外多糖的粘度为Xcc8004的63％。
用RT—PCR的方法对gum基因族gumA—gumB区段进行了转录分析，认为gumA—gumB基因区段具有两个可转录的区段ORF243和ORF233。为了进一步验证ORF243与色素有关，ORF233与胞外多糖的组分和粘度有关，我们成功的构建了这两个ORF的整合突变体TGL243#和TGL233#。结果表明整合突变TGL243#与插入突变体TGL243的的色素产量相一致，约为Xcc8004色素产量的34％。通过ncbi中的blast搜索，结果表明ORF243编码产物为一种转录调控蛋白，推测ORF243可能与色素的合成有关，而ORF233在ncbi中没有找到与之同源的基因编码产物。整合突变体TGL233#与插入突变体TGL233胞外多糖粘度相一致比Xcc8004低，约为Xcc8004的63％，推测ORF233与胞外多糖的粘度和组分有关。
The transcription of the gum operon in the Xanthomonas campestris pv. campestris 8004(Xcc 8004) and its TnSgusAS transposon insertion mutants of the gum genes was comparatively analyzed by the RT-PCR. These results showed that TnSgusAS insertion has the strong complete polarity. This result also verified that these three gum genes (gumK,gummL,gumM)form the second transcription in the gum gene operon.
The insertion sites of Tn5gusA5 in four mutants(TGL243,TGL14,TGL233 and TGL40) were located by methods of TAIL-PCR and sequencing. Biological and rheological characters were deterinated and analysised. The results showed the production of pigment in TGL243 were much lower than that in Xrc8004.lt is about 34% of Xcc8004; Production of pigment in TGL40 were much higher than that in Xcc8004.It is about 130% of Xcc8004; The ingredient and viscosity of excellular poysacchride (EPS) were changed in TGL233.The production of viscosity in TGL233 were 63% of Xcc8004.
The transcription of gap between gumA and gumB has been analysised and two transcriptional units(ORF243 and ORF233) has been found .
For further study, we constructed tow intergrated mutants of ORF243 and ORF233 successfully, named TGL243# and TGL233#. The pigment production of TGL243# like TGL243 were much lower than that of Xcc8004. The viscosity production of TGL233# were 63%.By the searching of ncbi blast, the results revealed that the deduced product encoded by ORF243 are predicated transcriptional regulators. So, we conclude ORF243 may be relevant to pigment production, ORF243 may be relevant to viscosity production and ingredient of EPS.
pv.campestris)8004(Xcc8004)及其Tn5gusA5转座子插入gum基因的突变体的gum基因操纵子的转录进行了比较分析。结果表明Tn5gusA5的插入具有很强的完全极性效应，并进一步证实gumK，gumL和gumM基因构成了gum基因操纵子中第二个转录本。
通过TAIL—PCR和测序方法对转座子Tn5gusA5插入突变体TGL243，TGL14，TGL233和TGL40中转座子Tn5gusA5在Xcc8004基因组中的位置进行了定位。并对这四株突变体的一些生物学特性和流变学特性进行了测定和分析，结果表明突变体TGL243色素产量约为Xcc8004色素产量的34％，TGL40约为Xcc8004的130％。TGL233胞外多糖的组分和粘度与Xcc8004不同，其胞外多糖的粘度为Xcc8004的63％。
用RT—PCR的方法对gum基因族gumA—gumB区段进行了转录分析，认为gumA—gumB基因区段具有两个可转录的区段ORF243和ORF233。为了进一步验证ORF243与色素有关，ORF233与胞外多糖的组分和粘度有关，我们成功的构建了这两个ORF的整合突变体TGL243#和TGL233#。结果表明整合突变TGL243#与插入突变体TGL243的的色素产量相一致，约为Xcc8004色素产量的34％。通过ncbi中的blast搜索，结果表明ORF243编码产物为一种转录调控蛋白，推测ORF243可能与色素的合成有关，而ORF233在ncbi中没有找到与之同源的基因编码产物。整合突变体TGL233#与插入突变体TGL233胞外多糖粘度相一致比Xcc8004低，约为Xcc8004的63％，推测ORF233与胞外多糖的粘度和组分有关。
The transcription of the gum operon in the Xanthomonas campestris pv. campestris 8004(Xcc 8004) and its TnSgusAS transposon insertion mutants of the gum genes was comparatively analyzed by the RT-PCR. These results showed that TnSgusAS insertion has the strong complete polarity. This result also verified that these three gum genes (gumK,gummL,gumM)form the second transcription in the gum gene operon.
The insertion sites of Tn5gusA5 in four mutants(TGL243,TGL14,TGL233 and TGL40) were located by methods of TAIL-PCR and sequencing. Biological and rheological characters were deterinated and analysised. The results showed the production of pigment in TGL243 were much lower than that in Xrc8004.lt is about 34% of Xcc8004; Production of pigment in TGL40 were much higher than that in Xcc8004.It is about 130% of Xcc8004; The ingredient and viscosity of excellular poysacchride (EPS) were changed in TGL233.The production of viscosity in TGL233 were 63% of Xcc8004.
The transcription of gap between gumA and gumB has been analysised and two transcriptional units(ORF243 and ORF233) has been found .
For further study, we constructed tow intergrated mutants of ORF243 and ORF233 successfully, named TGL243# and TGL233#. The pigment production of TGL243# like TGL243 were much lower than that of Xcc8004. The viscosity production of TGL233# were 63%.By the searching of ncbi blast, the results revealed that the deduced product encoded by ORF243 are predicated transcriptional regulators. So, we conclude ORF243 may be relevant to pigment production, ORF243 may be relevant to viscosity production and ingredient of EPS.
引文
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