【摘要】 wxocA、wxocB、wxocD、wxocE和wzt是水稻条斑病菌(Xanthom onas oryzaepv.oryzicola,Xooc)的脂多糖(lipopo-lysaccharide,LPS)合成基因,avrXa3是水稻白叶枯病菌(Xanthom onas oryzaepv.oryzae,Xoo)的一个无毒基因。利用pBCSK(-)和pKNG101两个质粒的复制起点不被水稻黄单胞菌识别的特点,把wxocA、wxocD、wxocE和wzt的中心区域克隆在pBCSK(-)上,获得突变转化单元pBCSK∷ΔwxocA、pBCSK∷ΔwxocD、pBCSK∷ΔwxocE和pBCSK∷Δwzt。把wxocB和avrXa3基因的旁侧序列和一个氯霉素抗性基因cm构建在pKNG101上,获得突变转化单元pKNG101∷ΔwxocB和pKNG101∷Δavr。以电转化方式把这些LPS合成基因突变转化单元DNA转入Xooc菌株RS105,把无毒基因突变转化单元转入Xoo菌株PXO99,通过同源重组分别获得了RS105中5个lps相应基因突变体MwxocA、MwxocB、MwxocD、MwxocE、Mwzt和一个无毒基因的突变体PXO99Δavr。SDS-PAGE分析发现,lps突变体的O抗原或核心寡糖的合成被破坏。致病性分析结果显示,基因wxocB、wxocE和wzt与致病性有关,前两基因的突变导致细菌完全失去毒性,而后一基因的突变导致细菌部分丧失毒性。与PXO99相比无毒基因突变体PXO99Δavr改变了原有的毒性表型,在IRBB50(Xa4/xa5)和Asom inori(Xa17)上由较感转变为较抗表型。因此,本试验证明,以pBCSK(-)和pKNG101为载体敲除水稻条斑病菌和白叶枯病菌致病相关基因是可行的。更多还原

【Abstract】 wxocA,wxocB,wxocD,wxocE and wzt are genes for lipopolysaccharide(LPS)biosynthesis in Xanthomonas oryzae pv.oryzicola(Xooc),and avrXa3 is an avirulence(avr)gene in Xanthomonas oryzae pv.oryzae(Xoo).Plasmids pBCSK(-)and pKNG101 with replicon of ColE1 and R6K,respectively,were used to construct gene knock-out transformation units.Transformation units of pBCSK∷ΔwxocA,pBCSK∷ΔwxocD,pBCSK∷ΔwxocE and PBCSK∷Δwzt to produce Xooc strain PS105 corresponding gene mutants were constructed with two fragments:central portion of target gene and pBCSK(-) vector.Transformation units of pKNG101∷ΔwxocB and pKNG101∷Δavr to produce wxocB mutant of Xooc strain RS105 and avirulence gene mutant in Xoo strain PXO99 were constructed with four fragments :pKNG101 vector,two flanking sequences of corresponding gene,and cm gene.Transformation was conducted by electroporation to yield 5 lps gene mutants(MwxocA,MwxocB,MwxocD,MwxocE,Mwzt)and 1 avr gene mutant(PXO99Δavr),and homologous recombinations were confirmed by Southern blot.The analysis of LPS by SDS-PAGE showed that all the lps gene mutants had defects in O-antigen biosynthesis.Virulence tests indicated that pathogenicity of MwxocB and MwxocE were completely lost and Mwzt was reduced.And PXO99Δavr showed less virulence on IRBB50(Xa4/xa5)and Asominori(Xa17).Therefore,the vectors pBCSK(-)and pKNG101 were first confirmed to be successfully used to knock-out genes in Xooc and Xoo and be helpful to identify the functions of pathogenicity-related genes in Xooc and Xoo.更多还原