水稻基腐病菌HrpX/HrpY在致病性中的功能分析

doi:10.3864/j.issn.0578-1752.2014.04.007

摘要/Abstract

摘要： 【目的】由Dickeya zeae引起的水稻基腐病是水稻上重要细菌病害之一，迄今对该病原细菌的致病性调控研究尚不完善。论文旨在分析水稻基腐病菌（Dickeya zeae）双组分调控系统HrpX/HrpY的序列特征，研究该系统在水稻基腐细菌中的功能作用以及与下游基因的调控关系。【方法】对HrpX/HrpY序列进行生物信息学分析，构建带有反向筛选标记基因scaB的自杀重组质粒pKNG-ΔhrpX和pKNG-ΔhrpY，通过三亲转化方法分别将重组质粒导入野生型菌株EC1中，通过2次等位基因同源重组筛选获得基因缺失突变体ΔhrpX和ΔhrpY；比较突变体与野生菌的生长速率、胞外酶活性、毒素活性、运动性、菌膜形成能力，以及对水稻的致病性和对烟草的过敏性反应（HR）；进一步提取细菌总RNA，采用实时荧光定量PCR研究HrpX/HrpY双组分系统对下游基因表达量的影响。【结果】水稻基腐病菌EC1基因组中，hrpX和hrpY均为单拷贝，hrpX编码区长1 473 bp，编码490个氨基酸，hrpY长642 bp，编码213个氨基酸，hrpX和hrpY两者之间相隔32 bp，具有相同的转录方向，在功能上具有相关性。其中，HrpX是一个结合在膜上的组氨酸蛋白激酶，HrpY是存在细胞质中的效应调节蛋白，HrpX/HrpY两者构成双组分系统，并对下游hrp基因的表达具有调控作用。进一步通过遗传操作手段，成功构建了基因缺失突变体ΔhrpX和ΔhrpY，表型测定结果表明，突变体ΔhrpX和ΔhrpY的运动性减弱、菌膜形成能力降低、对水稻种子萌发的抑制作用减弱，且降低了对水稻的致病力，但ΔhrpX和ΔhrpY的生长速率、胞外酶和毒素的活性变化不明显，也不影响其在烟草上的HR。实时荧光定量PCR结果显示，突变体菌株ΔhrpX和ΔhrpY相对野生菌EC1表达量明显下降的基因有hrpA、hrpF和hrpN，而对毒素合成基因zmsA表达量的差异不明显，HrpX/HrpY双组分系统对hrp基因簇中大部分下游基因都有正调控作用，且HrpY的调控作用更加明显。【结论】HrpX/HrpY是水稻基腐病细菌中的一个双组分系统，调节病原细菌的运动性、菌膜的形成和病菌的致病力，正向调控一些下游hrp基因的表达。

关键词: 水稻基腐病菌 , HrpX , HrpY , 运动性 , 致病性

Abstract: 【Objective】Rice bacterial foot rot caused by Dickeya zeae is one of the important bacterial diseases on rice. The virulence and regulation mechanism of D. zeae are imperfect at present. This paper aims to analyze the sequence characteristics of HrpX/HrpY, a two-component regulatory system (TCS) in D. zeae, and study on the roles of this system in D. zeae virulence and the relationships between HrpX/HrpY and its downstream genes.【Method】The sequences of HrpX/HrpY were analyzed by bioinformatics, a suicide recombinant plasmids pKNG-ΔhrpX and pKNG-ΔhrpY with reverse selection marker gene scaB were constructed, then transferred into wild strain EC1, respectively, by triparental mating, so the gene deletion mutants ΔhrpX and ΔhrpY were constructed after two alleles homologous recombination screening. The biological characteristics such as growth rate, extracellular enzyme, toxin, motility, biofilm, virulence on rice and HR on tobacco were compared and analyzed. In addition, bacterial total RNA was extracted, and real-time quantitative PCR was carried out so as to assess the expression of downstream genes of HrpX/HrpY.【Result】In the genome of D. zeae strain EC1, hrpX and hrpY were single copy. The coding region of hrpX was 1 473 bp, which encoding 490 amino acids, while hrpY with the size of 642 bp encodes 213 amino acids. There were only 32 base pairs between hrpX and hrpY. They were in the same transcriptional direction, and had functional relationship. HrpX was a histidine protein kinase combined with the bacterial membrane and HrpY was a response regulator in the cytoplasm. They constitute a two-component system and regulate the downstream genes expression of hrp. Further by genetic manipulation, two gene deletion mutants ΔhrpX and ΔhrpY were constructed successfully. The results of phenotypic determination showed that ΔhrpX and ΔhrpY lost motility, could not form pellicle (biofilm), weakened the inhibitory effect on rice and seed germination. Furthermore, mutants virulence tests showed that the symptoms alleviated than the wild strain EC1 did when the bacterial suspensions were poured into the root of rice plant. There were no significant differences between mutants and EC1 in growth rate, activities of extracellular enzyme and toxin production, they could also induce HR on tobacco. Real-time fluorescent quantitative PCR results showed that HrpX/HrpY regulated positively some downstream hrp genes, and the expression quantity of hrpA, hrpF and hrpN decreased obviously. Further analysis found that the regulation effect of HrpY was more significant than HrpX. 【Conclusion】HrpX/HrpY is a two-component regulatory system in D. zeae. It regulates bacterial motility, biofilm, and virulence, and regulates the expression of downstream hrp genes positively.

Key words: Dickeya zeae , HrpX , HrpY , motility , virulence

陈雪凤, 魏楚丹, 张庆, 刘琼光. 水稻基腐病菌HrpX/HrpY在致病性中的功能分析[J]. 中国农业科学, 2014, 47(4): 675-684.

CHEN Xue-Feng, WEI Chu-Dan, ZHANG Qing, LIU Qiong-Guang. Functional Analysis of HrpX/HrpY in Dickeya zeae Virulence[J]. Scientia Agricultura Sinica, 2014, 47(4): 675-684.

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