盐单胞菌DSM 16354<sup>T</sup>中新型耐盐基因的克隆及解析<sup>*</sup>

doi:10.13523/j.cb.2107020

中国生物工程杂志

2022, Vol. 42

Issue (3): 27-37 DOI: 10.13523/j.cb.2107020

研究报告

盐单胞菌DSM 16354^T中新型耐盐基因的克隆及解析^*

贾桂燕¹,王永杰¹,陈志康¹,陈星¹,殷奎德¹,李雯²,王艳红^1,^3,^**(

)

1 黑龙江八一农垦大学生命科学技术学院大庆 163319
2 黑龙江珍宝岛湿地国家级自然保护区管理局虎林 158419
3 黑龙江省寒区环境微生物与农业废弃物资源化利用重点实验室大庆 163319

Cloning and Analysis of Novel Functional Genes in Halomonas alkaliphila DSM 16354 ^T

JIA Gui-yan¹,WANG Yong-jie¹,CHEN Zhi-kang¹,CHEN Xing¹,YIN Kui-de¹,LI Wen²,WANG Yan-hong^1,^3,^**(

)

1 College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163319, China
2 Heilongjiang Zhenbao Island Wetland National Nature Reserve Administration, Hulin 158419, China
3 Heilongjiang Provincial Key Laboratory of Environmental Microbiology and Recycling of Argo-weast in Cold Region, Daqing 163319, China

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摘要：

目的: 嗜盐菌分布广泛且适应能力极强,为加强对嗜盐菌耐盐机制的探索与研究,从盐单胞菌Halomonas alkaliphila DSM 16354^T中筛选出潜在的与耐盐有关的基因,对其进行生物信息学分析,并验证相关蛋白的生理功能。方法: 利用基因文库筛选和功能互补相结合的方法,通过与大肠杆菌(Escherichia coli)盐敏感缺陷株KNabc(ΔnhaA、ΔnhaB、ΔchaA)的耐盐功能互补实验,筛选出具有耐盐功能的蛋白编码基因,并通过荧光猝灭恢复实验测定蛋白的逆向转运活性以及底物亲和力。结果: 筛选得到两个具有耐盐功能的蛋白编码基因,生物信息学分析结果表明该基因编码来自于DUF1538(domain of unknown function with No.1538 family)家族功能未知的膜蛋白,分别命名为duf1和duf2。系统发育树分析结果表明,来自盐单胞菌DSM 16354^T中的DUF1、DUF2属于一个独立的分支,预测这两个蛋白可能是DUF1538家族转运蛋白的新成员。对DUF1和DUF2的生理功能进行分析,发现duf1和duf2单独表达时均不具有耐盐碱能力,而共同表达时则表现出显著的耐盐碱功能,表明DUF1和DUF2两个亚基共同支持了蛋白的耐盐碱功能。蛋白的逆向转运测定活性结果表明双组份蛋白DUF1-2具有Na⁺(Li⁺、K⁺)/H⁺逆向转运活性。结论: 筛选得到的基因duf1和duf2共同表达时具有盐碱耐受功能以及逆向转运蛋白活性,这为筛选出新的DUF1538家族转运蛋白基因和进一步探究DUF1538家族转运蛋白功能奠定了基础。

关键词： 盐单胞菌; DUF1538家族; 钠/氢逆向转运蛋白; 克隆; 生物信息学

Abstract:

Objective: Halophilic bacteria are widely distributed and have strong adaptability, but the research on their mechanism is not deep enough. In order to strengthen the exploration and research on the mechanism of salt tolerance of halophilic bacteria, potential genes related to salt tolerance were screened from H. alkaliphila DSM 16354 ^T, informatics analysis was carried out, and the physiological functions of the related proteins were verified. Methods: Using the combination of gene library screening and functional complementarity, through the salt tolerance functional complementarity experiment with E.coli salt sensitive defective strain KNabc (ΔnhaA、ΔnhaB、ΔchaA), the protein coding gene with salt tolerance function was screened, and then the reverse transport activity and substrate affinity of the protein were determined by fluorescence quenching recovery experiment. Results: Two protein coding genes with salt tolerance function were screened. Bioinformatics analysis showed that the gene encoded membrane proteins with unknown function from DUF1538(domain of unknown function with No.1538 family), named DUF1 and DUF2, respectively. Phylogenetic tree analysis showed that DUF1 and DUF2 from H. alkaliphila DSM 16354 ^T belonged to an independent branch. It was predicted that these two proteins may be new members of DUF1538 family transporters. The physiological functions of DUF1 and DUF2 were analyzed which did not have salt tolerance when expressed alone, but showed significant salt tolerance when expressed together, indicating that duf1 and duf2 subunits jointly supported the salt tolerance function of the protein. The activity of protein DUF1-2 reverse transport assay showed that the two-component protein DUF1-2 had Na ⁺ (Li⁺、K⁺)/H⁺ antiporter activity. Conclusion: The selected duf1 and duf2 transporters had salt-alkali tolerance function and antiporter activity when they were co-expressed. This laid a foundation for screening new DUF1538 family transporter genes and further exploring the function of DUF1538 family transporters.

Key words: H. alkaliphila DUF1538 family transporter Na⁺/H⁺ antiporter Clone Bioinformatics

收稿日期: 2021-07-06 出版日期: 2022-04-07

ZTFLH:

Q782

基金资助: * 国家自然科学基金(31771692)

通讯作者: 王艳红 E-mail: wyh_03@126.com

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引用本文:

贾桂燕,王永杰,陈志康,陈星,殷奎德,李雯,王艳红. 盐单胞菌DSM 16354^T中新型耐盐基因的克隆及解析^*[J]. 中国生物工程杂志, 2022, 42(3): 27-37.

JIA Gui-yan,WANG Yong-jie,CHEN Zhi-kang,CHEN Xing,YIN Kui-de,LI Wen,WANG Yan-hong. Cloning and Analysis of Novel Functional Genes in Halomonas alkaliphila DSM 16354 ^T. China Biotechnology, 2022, 42(3): 27-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2107020 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I3/27

表1 引物序列

图1 重组质粒中序列分析结果

图2 系统进化发育树的构建

图3 蛋白的拓扑结构

图4 重组菌株的耐盐碱测试

图5 钠/氢逆向转运蛋白活性的测定

图6 蛋白活性的pH轮廓

图7 DUF1-2 对Na+、Li+、K+的K0.5值的计算

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