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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2015, Vol. 41 Issue (1): 56-63    DOI: 10.3785/j.issn.1008-9209.2014.03.122
生物科学与技术     
组氨酸激酶基因barA在水生拉恩菌中的生防调控功能
肖凤虎1 , 张蕾2 , 谢镇1, 郭岩彬3 , 陈敏文1, 王勇军1,4*
1.浙江农林大学林业与生物技术学院,浙江 临安311300;2.浙江科技学院,杭州310023;3.中国农业大学资源与环境学院,北京100094;4.浙江农林大学生物农药高效制备技术国家地方联合工程实验室,浙江 临安311300
Regulatory function of histidine kinase sensor encoding gene barA in bio-control effect of Rahnella aquatilis
Xiao Fenghu1, Zhang Lei2, Xie Zhen1, Guo Yanbin3, Chen Minwen1, Wang Yongjun1,4*
(1. School of Forestry and Bio-technology, Zhejiang Agricultural and Forestry University, Lin’an 311300, Zhejiang, China; 2. Zhejiang University of Science and Technology, Hangzhou 310023, China; 3. College of Resources and Environmental Sciences, China Agricultural University, Beijing 100094, China; 4. National and Provincial Joint Engineering Laboratory of Bio-pesticide Preparation, Zhejiang Agricultural and Forestry University, Lin’an 311300, Zhejiang, China)
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摘要： 为探究水生拉恩菌(Rahnella aquatilis)HX2的生防性状表现机制,利用转座子mini-Tn5对HX2菌株进行随机突变,筛选获得1个对葡萄根癌菌K308(Agrobacterium vitis K308)拮抗活性减弱的突变体,并确定该插入位点的基因为双组分调控系统中的组氨酸激酶基因barA。该基因编码的蛋白含有组氨酸激酶结构域HAMP(histidine kinases, adenylyl cyclases, methyl binding proteins, phosphatases)、组氨酸磷酸传递结构域HisKA(histidine kinase acceptor)、C-端催化ATP结合结构域HATPase_C(histidine associated ATPase C terminal)、磷酸接受结构域REC(receiver domain)和含组氨酸的磷酸转移结构域HPT(histidine phosphotransferase domain),是一种跨膜的杂合组氨酸激酶蛋白。通过双交换突变,获得了barA缺失突变体并构建了互补菌株。通过比较分析发现,barA基因缺失后,细菌HX2生物膜形成能力显著提高,但细菌游动和涌动能力降低,以及对葡萄根癌病的生防效果从野生型的86.2%降低至26.7%,构建的互补菌株能够恢复突变菌株在该研究中的所有测定性状。由此推测,组氨酸激酶基因barA是细菌HX2表现生防性状的一个重要调控基因,为生防细菌HX2在植物病害防治上提供理论基础。
Abstract: Rahnella aquatilis HX2, which was isolated from the vineyard soil, is a gram-negative, plant growth-promoting rhizobacteria. Previous results showed that R. aquatilis HX2 has significant antagonistic effect on certain pathogenic bacteria and fungi including Agrobacterium tumefaciens, Xanthomonas oryzae, Fusarium oxysporum, Botrytis cinerea, Altemaria solani, etc, and exhibited the potential biocontrol value against rice sheath blight and crown gall of grapevine and sunflower. The completed genomic DNA sequence of R. aquatilis HX2 has been finished. For further discovery of the regulatory systems which regulate its biocontrol and physiological traits, random mutagenesis based on mini-Tn5 transposon was used to investigate the regulatory genes. The candidated genes were focused on the regulatory function in biocontrol-related physiological traits and biocontrol effects. Consequently, a mutant TR57 which had less antagonitic effect against the plant pathogen Agrobacterium vitis K308 was obtained after the random mutagenesis based on mini-Tn5 transposon and antagonitic assay. The DNA sequence flanking the inserted mini-Tn5 transposon was verified as a BarA-liked histidine kinase gene. The putative BarA protein in R. aquatilis HX2 contains HAMP domain, HisKA domain, HATPase_C domain, REC domain and HPT domain. BarA has been reported as the sensor protein of a two-component regulatory system BarA/UvrY in many bacteria, such as Escherichia coli, Pseudomonas spp. and the BarA/UvrY was known as a global regulatory functioning in bacterial survival under the circumstance of pH value and nutrition change. For further investigation of BarA in R. aquatilis HX2, null barA mutant was constructed based on homologous recombination. A vector pSRΔbarA was constructed after inserting the flanking region of barA loci into the suicide vector pSR47S, and transformed into E. coli DH5α (λ-pir). The triparental mating strategy was used to transfer the vector pSRΔbarA into R. aquatilis HX2. After the two-step homologous recombination, the null barA mutant MR57 was obtained consequently. Meanwhile, the complemented vector pRKbarA was constructed after inserting the barA operon into the shuttle vector pRK415G, and then was transformed into MR57. The biocontrol-related physiological traits and biocontrol effect of R. aquatilis HX2 and its derivative strains were compared to valuate the barA regulation function. The experimental results indicated that the mutagenesis of barA caused higher bacterial biofilm formation ability, less swimming and swarming ability. The biocontrol efficiency of barA mutant against A. vitis K308 on grape plants decreased to 26.7%, comparing to 86.2% of the wild type strain. Moreover, the complemented strain could recover all the measured biological characters and bio-control efficiency. Therefore, it was supposed that the histidine kinase gene barA plays the key role in biocontrol function of R. aquatilis HX2. In summary, barA is firstly found as a regulation gene functioning in bacterial biocontrol effect in this study. The results also give us the indication that the modification of bacterial two-component regulation systems would be helpful to facilitate the application of biocontrol bacteria.
出版日期: 2015-01-20
CLC:  Q 754  
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肖凤虎
张蕾
谢镇
郭岩彬
陈敏文
王勇军

引用本文:

肖凤虎,张蕾,谢镇, 郭岩彬,陈敏文, 王勇军. 组氨酸激酶基因barA在水生拉恩菌中的生防调控功能[J]. 浙江大学学报(农业与生命科学版), 2015, 41(1): 56-63.

Xiao Fenghu, Zhang Lei, Xie Zhen, Guo Yanbin, Chen Minwen, Wang Yongjun. Regulatory function of histidine kinase sensor encoding gene barA in bio-control effect of Rahnella aquatilis. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(1): 56-63.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2014.03.122    或    http://www.zjujournals.com/agr/CN/Y2015/V41/I1/56

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