假单胞菌HT66的PhzI-PhzR调控系统的功能研究

doi:10.3969/J.ISSN.1671-9964.2016.01.002

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摘要

绿针假单胞菌(Pseudomonas chlororaphis)HT66是一株分泌高水平吩嗪-1-甲酰胺(phenazine-1-carboxamide, 简称PCN)的植物根际促生细菌。通过全基因组测序与分析, 发现phz基因簇上游存在着PhzI-PhzR双元调控系统。显色实验表明, 野生株信号分子抽提物不能使指示菌紫色杆菌(Chromobacterium violaceum)CV026显紫色, 但能使指示菌根癌农杆菌(Agrobacterium tumefaciens)NTL4显蓝色。研究采用基因无痕敲除方法构建了突变株ΔphzⅠ、ΔphzR;与野生株相比, ΔphzⅠ突变株丧失了PCN合成能力, 对终极腐霉的抑制作用明显下降;而且突变株ΔphzⅠ的信号分子抽提物也不能使指示菌NTL4菌显色。由此可见, 菌株HT66以高丝氨酸内酯作为群体感应的信号分子, 且吩嗪的生物合成受到了PhzI-PhzR的严格调控。进一步形态观察表明, 突变株ΔphzⅠ的菌落颜色变为乳白色, 鞭毛泳动性与野生株相比大大降低, 但其群体从动性变化不显著。

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	欧阳夷
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	张雪洪

关键词 ：绿针假单胞菌, PhzI-PhzR双元调控系统, 群体感应

Abstract：

Pseudomonas chlororaphis HT66 is a significant growth-promoting rhizobacteriaum in plants.It is reportedly the highest phenazine-1-carboxamide(PCN)producing wild type microbe studied thus far. Based on the whole-genome sequence analysis, we find that two genes phzⅠ and phzR are located just upstream of the phenazine biosynthesis gene cluster.Plate assays with Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 were used to analyze the signals of strain HT66.With the addition of the fermentation broth extracts, strain NTL4 produced blue pigment, but the CV026 did not produce purple pigment.Then, using a gene knock-out method, we constructed ΔphzⅠ and ΔphzR HT66 mutants.The results show that both the ΔphzⅠ and ΔphzR mutants are unable to produce PCN.And the plate assay of ΔphzⅠ culture extracts shows no blue pigment is produced by strain NTL4.It suggests that phenazine biosynthesis is regulated by the quorum sensing system and AHL signals in strain HT66.Additionally, the ΔphzⅠ and ΔphzR mutants have some changes in the morphology of colonies and antifungal activity in vitro.Moreover, compared with wild type, the swimming motility of ΔphzⅠ mutant decreases greatly, while the swarming motility has no significant change.

Key words： Pseudomonas chlororaphis PhzI-PhzR system quorum sensing

收稿日期: 2015-03-25 出版日期: 2016-03-08

基金资助:国家自然科学基(31270084);国家“973”计划项目(2012CB721005)

作者简介:

作者简介: 欧阳夷(1990-), 女, 研究方向:微生物资源与代谢, E-mail:oyy1028@163.com;

通讯作者: 彭华松(1974-), 男, 副教授, 研究方向:微生物资源与代谢, E-mail:hspeng@sjtu.edu.cn

引用本文:

欧阳夷, 彭华松, 王威, 胡洪波, 张雪洪. 假单胞菌HT66的PhzI-PhzR调控系统的功能研究[J]. 上海交通大学学报(农业科学版), 2016, 34(1): 5-10.
Yi OUYANG, Hua-song PENG, Wei WANG, Hong-bo HU, Xue-hong ZHANG. Function of thePhzI-PhzR System in Pseudomonas chlororaphis HT66. Journal of Shanghai Jiaotong University (Agricultural Sciences), 2016, 34(1): 5-10.

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http://www.qk.sjtu.edu.cn/jsjtua/CN/10.3969/J.ISSN.1671-9964.2016.01.002 或 http://www.qk.sjtu.edu.cn/jsjtua/CN/Y2016/V34/I1/5

Tab.1 引物名称及序列

Fig.1 敲除株ΔphzⅠ、ΔphzR的PCR验证电泳图谱

注: a图, M为DNA marker, 1为突变株ΔphzⅠ, 2为野生型HT66;b图, M为DNA marker, 1与2均为突变株ΔphzR, 3为野生型HT66

Fig.2 假单胞菌HT66中野生株和敲除株ΔphzⅠ、ΔphzR的PCN产量

Fig.3 假单胞菌HT66中信号分子的NTL4平板检测

注: 1: HT66;2:ΔphzⅠ 突变抹;3:乙腈

Fig.4 鞭毛泳动性与群体从动性实验

Fig.5 假单胞菌HT66野生株与ΔphzⅠ、ΔphzR菌落形态的比较

Fig.6 假单胞菌HT66的野生株与ΔphzⅠ、ΔphzR突变株的抑菌活性

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