野油菜黄单胞菌野油菜致病型胞外多糖产生有关的一基因簇的鉴定和分析
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摘要
本室原已从Xcc野生型菌株8004克隆到含有EPS产生相关基因的9.4kbHindⅢ DNA片段，本研究进一步构建了这一片段的6种限制性内切酶酶切图谱，通过转座子Tn5gusA5插入诱变这一克隆片段和标记置换(marker exchange)构建突变体，发现其中连续的7kb区域中含有一个与EPS产生有关的基因簇，由于未能获得转座子插入及实质，还不清楚于另一端的余下的2kb区域是否与EPS产生有关。将含有这9.4kb DNA片段的重组质粒导入菌株8004，能提高EPS的产量2.8倍。
本研究对以上9.4kbDNA中的“1.9”kb EcoR1 片段进行了测序分析，测序分析结果表明这一片段实际长度为1880bp。进一步对这1880bp DNA进行分析，其中含有一个完整的开放阅读框架(ORF2)和一个不完整的开往阅滨框架(ORF1)，ORF1和ORF2的推断性编码产物分别与已报道的GumA和GumB蛋白在氨基酸水平上具有100％的同源性。GumA是一种作用范围较广的调控蛋白，被称为整合寄主因子(IHFα)，在大肠杆菌中是由himA基因编码的。GmB可能与糖核苷聚合或EPs输出有关。
本工作还利用所构建的EPS-突变体探讨了EPS对Xcc致病性的影响，结果表明，EPS的产量与致病性呈正相关，按种浓度的大小直接影响到致能力。通过电镜观察还发现，EPS对Xcc从气孔入侵寄主能力具有重要作用。
A 9.4kb HindIII-DNA fragment harbouring a gen (or gens) inolved in EPS production was cloned preiously from Xcc wild-type strain 8004 in our laboratory. In this study, a restriction nap with 6 enzymes of the fragment was constructed. By inserton mutagenesis with transposon Tn5 A5 of the fragment and construction of mutants by marker exchange it was revealed that there is a gene cluster involved in EPS production spanning 7 kb continuous region within the 9.4kb DNA fragment. It is not known if the rest 2 kb region is involved in EPS production, as no insertion or mutation was obtained within this region.
Introduction of the recombinant plasmid harbouring the 9. 4kb Hind HI DNA fragment into the strain 8004 resulted in 2.8-fold increase of EPS production.
A"1.9kb"EcoRl fragment within the above mentioned 9.4kb DMA was sequenced and analyzed in this study. The results revealed that the fragment contains 1880bp nucleiotide with a complete open reading frame (0RF2) and an incomplete ORF(ORFl). The deduced products encoded by 0RF1 and 0RF2 show 100X indentity with the reported GumA and GumB proteins of Xec at amino acid level, respectively. The GumA is a regulator with extensive regulations, often referred to integration host factor alpha (IHFa )encoded by the himA gene in Escherichia coli. The GumB probably play a role in the polymerization of sugar nucleiotides or exportation of EPS.
This work studied also the effect of EPS in the pathogenicity of Xcc by inoculating the constructed EPS mutants(EPS-) on host plants.The results showed that EPS has an important role in the pathogenicity, the inoculation closes affect the symptom production. The results of scanning electron microscopy revealed that EPS might be important for infection of Xcc via stomata.
本研究对以上9.4kbDNA中的“1.9”kb EcoR1 片段进行了测序分析，测序分析结果表明这一片段实际长度为1880bp。进一步对这1880bp DNA进行分析，其中含有一个完整的开放阅读框架(ORF2)和一个不完整的开往阅滨框架(ORF1)，ORF1和ORF2的推断性编码产物分别与已报道的GumA和GumB蛋白在氨基酸水平上具有100％的同源性。GumA是一种作用范围较广的调控蛋白，被称为整合寄主因子(IHFα)，在大肠杆菌中是由himA基因编码的。GmB可能与糖核苷聚合或EPs输出有关。
本工作还利用所构建的EPS-突变体探讨了EPS对Xcc致病性的影响，结果表明，EPS的产量与致病性呈正相关，按种浓度的大小直接影响到致能力。通过电镜观察还发现，EPS对Xcc从气孔入侵寄主能力具有重要作用。
A 9.4kb HindIII-DNA fragment harbouring a gen (or gens) inolved in EPS production was cloned preiously from Xcc wild-type strain 8004 in our laboratory. In this study, a restriction nap with 6 enzymes of the fragment was constructed. By inserton mutagenesis with transposon Tn5 A5 of the fragment and construction of mutants by marker exchange it was revealed that there is a gene cluster involved in EPS production spanning 7 kb continuous region within the 9.4kb DNA fragment. It is not known if the rest 2 kb region is involved in EPS production, as no insertion or mutation was obtained within this region.
Introduction of the recombinant plasmid harbouring the 9. 4kb Hind HI DNA fragment into the strain 8004 resulted in 2.8-fold increase of EPS production.
A"1.9kb"EcoRl fragment within the above mentioned 9.4kb DMA was sequenced and analyzed in this study. The results revealed that the fragment contains 1880bp nucleiotide with a complete open reading frame (0RF2) and an incomplete ORF(ORFl). The deduced products encoded by 0RF1 and 0RF2 show 100X indentity with the reported GumA and GumB proteins of Xec at amino acid level, respectively. The GumA is a regulator with extensive regulations, often referred to integration host factor alpha (IHFa )encoded by the himA gene in Escherichia coli. The GumB probably play a role in the polymerization of sugar nucleiotides or exportation of EPS.
This work studied also the effect of EPS in the pathogenicity of Xcc by inoculating the constructed EPS mutants(EPS-) on host plants.The results showed that EPS has an important role in the pathogenicity, the inoculation closes affect the symptom production. The results of scanning electron microscopy revealed that EPS might be important for infection of Xcc via stomata.
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