核桃细菌性黑斑病菌rpfG基因的功能分析

Functional analysis of rpfG gene in Xanthomonas arboricola pv.juglandis

【目的】本研究旨在揭示核桃细菌性黑斑病菌(Xanthomonas arboricola pv.juglandis,Xaj)DW3F3中rpfG基因的生物学功能,从而为核桃细菌性黑斑病防治药剂的开发提供作用靶点。【方法】以野油菜黄单胞菌(Xanthomonas campestris pv.campestris,Xcc)8004菌株以及水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)PXO99A的rpfG基因为模板序列,对Xaj野生型菌株DW3F3的基因组序列进行检索。利用同源重组技术,对Xaj中rpfG基因进行敲除,并用生物化学方法对基因缺失菌株的相关毒力因子、抗逆性进行检测。【结果】通过同源比对,在XajDW3F3的基因组中发现了与XccrpfG、XoorpfG同源的基因,并成功获得rpfG的缺失突变株ΔrpfG。与野生型相比,突变株ΔrpfG的生物被膜形成能力仅为野生型XajDW3F3的44.58%;胞外多糖产量也由野生型的8.47 mg/mL降为5.23 mg/mL;ΔrpfG的絮凝活性增加,能使菌液变澄清;运动性实验显示ΔrpfG的运动直径比野生型增加了12.38%;胞外酶的分泌也发生了不同程度的改变,突变株分泌纤维素酶的能力极显著降低,淀粉酶活性有所提高,而分泌蛋白酶的能力未发生变化;此外rpfG缺失后,Xaj对逆境(盐、酸、SDS、硫酸铜)的耐受力降低。【结论】结果表明rpfG基因能影响核桃细菌性黑斑病菌的致病相关性状,并赋予了细菌一定的抗逆性。

[Objective]The purpose of this study was to reveal the biological function of rpfG gene in walnut bacterial black spot pathogen(Xanthomonas arboricola pv.juglandis)Xaj DW3F3,so as to provide a target for the development of prevention and treatment agents for walnut bacterial black spot.[Methods]The rpfG gene of Xanthomonas campestris pv.campestris 8004 and Xanthomonas campestris pv.campestris PXO99A were used as template sequences to search the genome sequence of DW3F3,a wild-type strain of Xaj.With homologous recombination technology to knock out XajrpfG,the virulence factors and stress resistance of the mutant strain were detected by biochemical methods.[Results]Homologous gene was identified in Xaj DW3F3 genome through sequence homology analysis.The deletion mutant of rpfG was successfully obtained.Compared with the wild type,the mutant reduced the production of biofilm and extracellular polysaccharides,the ability of biofilm formation mutant strains ΔrpfG was only about 44.58% of wild type,the yield of exopolysaccharide decreased from 8.47 mg/mL in wild type to 5.23 mg/mL in ΔrpfG;but ΔrpfG increased the motility and flocculation activity,motility experiments showed that the motility diameter of ΔrpfG increased 12.38% than the wild type.The secretion of extracellular enzymes also changed at different degrees:the ability to secrete cellulase was significantly decreased and amylase activity was increased,but the ability to secrete protease was not changed.What’s more,after the deletion of rpfG,the resistance of Xaj to adversity(NaCl,pH,SDS,CuSO4)was decreased.[Conclusion]The results showed that rpfG is an important pathogenic gene of walnut bacterial black spot pathogen,and endows bacteria with a certain degree of resistance to adversity.