欧美杨细菌性溃疡病菌hrcJ基因的功能分析  

Functional Analysis of hrc J Gene in Lonsdalea quercina subsp. populi

作　　者：李宾[1] 李爱宁[1] 魏强[1] 王海明 贺伟[1] 

机构地区：[1]北京林业大学林木有害生物防治北京市重点实验室,北京100083 [2]山东省菏泽市林业局,菏泽274000

出　　处：《林业科学》2015年第12期71-78,共8页Scientia Silvae Sinicae

基　　金：中央高校基本科研业务费专项资金资助(BLX2013015);林业公益性行业专项资助项目(201104054)

摘　　要：【目的】为了明确欧美杨细菌性溃疡病病原菌hrc J基因在致病过程中的作用,解析病原菌Ⅲ型分泌系统各基因的功能,为阐明病原菌的致病机制提供理论依据。【方法】利用同源重组原理,构建携带菌株N-5-1 hrc J基因的自杀载体p EX18Km-hrc J,运用两亲结合方法把自杀载体导入野生型N-5-1菌株中,在含有相应浓度的卡纳霉素和利福平抗生素的LB平板上筛选得到hrc J基因的插入突变体,并通过PCR和Southern杂交验证。为进一步验证p EX18Km-hrc J是否定点整合入hrc J基因,设计了2对引物组合进行PCR扩增。在室内生化培养箱内,分别以野生型菌株N-5-1、插入突变体菌株Δhrc J和互补载体菌株HBhrc J接种1年生107杨苗干,进行致病性测定。【结果】扩增出来的PCR片段大小均与预期一致,Southern杂交信号带大小与预期相同,表明自杀载体p EX18Kmhrc J正确地整合入目的基因,即hrc J基因由于发生同源交换而实现了插入突变。生物学特性测试表明:hrc J基因的插入突变体在1年生107杨苗干上的致病能力显著下降,野生型菌株N-5-1、插入突变体菌株Δhrc J和互补载体菌株HBhrc J的病情指数分别为84.4,31.1和66.7,并且丧失了在本氏烟草上的HR激发能力,功能互补突变体部分恢复至野生表型;生长能力测定显示,野生型菌株N-5-1、插入突变体Δhrc J和互补载体HBhrc J生长曲线无明显差异,在液体LB培养基中均可正常生长;通过结晶紫染色后,在培养Δhrc J、HBhrc J、野生菌株N-5-1的试管壁上均可以观察到紫色环状的生物膜形成,3种菌株的OD_(570)值相近;游动性测试表明,hrc J基因突变体的运动能力比野生菌株下降了21%。【结论】利用自杀载体p EX18Km可成功构建欧美杨细菌性溃疡病病原菌基因插入突变载体,为欧美杨细菌性溃疡病病菌的致病基因研究提供一种可选择的方法。欧美杨细菌性溃疡病菌hrc J基因与其对107杨的致病[ Objective] The poplar bacterial canker caused by Lonsdalea quercina subsp, populi is a disease that is a seriously harm to poplar industry. In order to clarify the role of the hrcJ gene playing in the pathogenesis of L. quercina subsp, populi, it＇ s necessary to reveal the function of each gene in type Ⅲ secretion system of the pathogen and provide a theoretical basis for elucidating the pathogenic mechnism of pathogens. [ Method ] The hrcJ inserted mutant was constructed by homologous recombination. A suicide vector, pEX18Km-hrcJ,that carries hrcJ gene of the pathogen strain N-5-1 was constructed. The suicide vector was introduced into the wild-type strain N-5-1 by using amphiphilic bonding method. The hrcJ gene insertion mutants were screened on LB plates containing the corresponding concentration of kanamycin and rifampicin. Then the hrcJ inserted mutant was verified by PCR and Southern blot. One-year-old seedling of Populus x euramericana cv. ＇74/76＇ were inoculated in an incubator, with strains of the wild-type, the hrcJ gene mutant, and the complemented mutant HBhrcJ respectively, for pathogenecity test. [ Result ] The PCR amplified fragment sizes are consistent with those expectations and Southern hybridization signal stripe size was the same as expected, showing that the suicide vector pEX18Km-hrcJ has been properly integrated into the target gene. That is the hrcJ gene has achieved insertion mutation due to the homologous exchange. Pathogenicity test on one-year-old poplar branches indicated that thehrcJ gene mutant was significantly less virulent than wild-type, while the complemented mutant HBhrcJ restored the virulence to the wild-type level. Growth analysis showed that growth capacity of hrcJ gene mutant had no significant change compared with that of wild-type, and the ability of biofihn fin＇mation evaluated by ODs7o was similar among strains N-5-1 , AhrcJ and HBhrcJ. Motility test showed that the motility of hrcJ gene mutant was decreased by 21 percent compared with that of wild-type. [

关 键 词：欧美杨细菌性溃疡病菌 hrc J Ⅲ型分泌系统 致病性 

分 类 号：S763.1[农业科学—森林保护学]