摘要
西瓜噬酸菌(Acidovorax citrulli, Ac)引起的细菌性果斑病(bacterial fruit blotch of watermelon, BFB)是一类为害葫芦科植物的重要细菌病害,在世界的大多数国家和地区都有分布,造成严重的经济影响。西瓜噬酸菌也是我国的检疫性有害生物,目前关于该病原物的研究主要集中在检测防治方面,但是关于它的致病机制以及侵染途径方面的研究相对较少。因此本研究的目的就是克隆致病相关基因,并对其进行功能分析。
利用转座子(Mini-Tn5)对Acidovorax citrulli strain xjL12 (Ac xjL12)进行转座诱变,构建了西瓜噬酸菌Ac xjL12的转座子插入文库。以接种哈密瓜幼苗的方法筛选得到两株株致病力完全丧失的突变株Δxj48,通过亚克隆的方法,鉴定了Δxj13、Δxj48的Mini-Tn5插入位点分别为pyrG基因、hpaP基因。
利用自杀载体pVIK112,应用两亲交配的方式,构建了插入失活突变体ΔpyrG和ΔhpaP,并对其功能进行了初步分析。研究表明ΔpyrG、ΔhpaP丧失了对哈密瓜幼苗的致病性,这个结果与Δxj13、Δxj48一致；通过基因互补,分别构建了互补菌株pyrG hb、hpaPhb,互补菌株都基本恢复了对哈密瓜子叶的致病性。分别将野生型、突变株以及互补菌株接种烟草,发现Δxj13、Δxj48、ΔpyrG、AhpaP、hpaPhb都丧失了引起过敏性反应的能力,而pyrGhb恢复引起过敏性反应的能力；随后还对突变体的其他表型进行了研究,发现基因pyrG、hpaP突变后影响了AcxjL12的游动性、生物膜的形成能力,还影响了病原菌的定殖能力；反转录差异显示hpaP基因的突变对hrcV基因的表达有影响,pyrG基因的突变对hisD基因的表达有影响；MMX培养基中添加组氨酸的生长曲线测定表明pyrG基因的突变影响西瓜噬酸菌对组氨酸的利用。证实了西瓜噬酸菌中的pyrG、hpaP基因与该细菌的致病力相关,在其生长过程和侵染过程中起着不可缺失的作用。
Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a threatening disease of watermelon, melon and other cucurbits, which have been reported in many countries and areas, and caused great economic losses. Acidovorax citrulli is a quarantine pest, until now most studies on this pathogen related to its detection and control, whereas few researches focused on its pathogenic mechanism and infextation way. Accordingly, this study aimed at cloning pathogenicity-related genes and analyzing their functions.
The mutant library of Acidovorax citrulli strain xjL12 was constructed by mating Mini-Tn5 transposon into Ac xjL12. Two mutantΔxj13、Δxj48, which lost panthogenicity were screened, the transposon insertion site were identified as pyrG, hpaP by sub-clone stategy.
With the help of suicide plasmid, disruption mutants ApyrG and AhpaP were constructed by a method of homologous recombination. The result showed that ApyrG, AhpaP lost pathogenicity to hami seedlings. Complement strains pyrG hb, hpaPhb generally recoverd pathogenicity; HR assays indicated strainsΔxj13,Δxj48,ΔpyrG,ΔhpaP and hpaPhb losted this ability, but strain pyrGhb regained it. Then, other phenotypes were studied, characterization of the mutants revealed that swimming motility, biofilm formation and growth in host plant demanded gene pyrG, hpaP; RT-PCR showed that the expression of hrcV gene needed hpaP gene and pyrG gene affected the expression of hisD gene; strainΔpyrG could not grow normally in the auxotrophic media (MMX) added histidine; These evidences in the stusy demonstrated that gene hpaP, pyrG is cirtical for the pathogenicity of Acidovorax citrulli, and plays an important role in the process of growth and infection.
引文
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