[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 × 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 the hrcJ 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 biofilm formation evaluated by OD570 was similar among strains N-5-1,ΔhrcJ and HBhrcJ. Motility test showed that the motility of hrcJ gene mutant was decreased by 21 percent compared with that of wild-type.[Conclusion]The suicide vector pEX18Km can be successfully used to construct pathogen gene insertion mutation in Lonsdalea quercina subsp. populi,and an alternative method is provided for pathogenic gene research of the pathogen. This result suggests that the hrcJ gene is required for pathogenicity of L. quercina on host plant poplar and hypersensitive reaction on non host plant tobacco. The hrcJ gene mutant of L. quercina did not affect growth of the pathogen and the formation of biofilm,but the obviously decreased motility.%【目的】为了明确欧美杨细菌性溃疡病病原菌 hrcJ 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 hrcJ基因的自杀载体 pEX18Km-hrcJ，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 hrcJ基因的插入突变体，并通过 PCR 和 Southern杂交验证。为进一步验证pEX18Km-hrcJ是否定点整合入hrcJ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ΔhrcJ 和互补载体菌株 HBhrcJ 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 pEX18Km-hrcJ正确地整合入目的基因，即 hrcJ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： hrcJ 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ΔhrcJ 和互补载体菌株 HBhrcJ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ΔhrcJ和互补载体 HBhrcJ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ΔhrcJ、HBhrcJ、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 OD570值相近；游动性测试表明，hrcJ 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 pEX18Km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hrcJ基因与其对107杨的致病性有关，能够激发非寄主植物烟草的过敏性反应；欧美杨细菌性溃疡病菌 hrcJ基因突变并未影响病原菌的生长和生物膜的形成，但其游动性减弱。
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