Cloning and expression of Actinokineospora alba DSM 45114T rpf gene and structure prediction of Rpf protein

doi:10.3969/j.issn.1000-1565.2022.04.011

Abstract

Abstract: Resuscitation-promoting factor(Rpf)is widely present in gram-positive bacteria with high G+C content and is related to the resuscitation of dormant cells. There are some differences in the types and quantities of Rpf among different bacteria. In order to understand the difference of rpf genes in different species, bioinformatics analysis was conducted on the amino acid sequence, hydrophobicity, signal peptide, phosphorylation site, tertiary structure and structural functional domain of three Rpf proteins in Actinokineospora alba DSM 45114^T. The specific primers for the three rpf genes of A. alba- DOI:10.3969/j.issn.1000-1565.2022.04.011白色放线动孢菌 DSM 45114^T rpf基因的克隆表达及结构预测张宇帆,刘菁菁,曹敏,刘彤彤,陈雪,张秀敏(河北大学生命科学学院,生命科学与绿色发展研究院,河北省微生物育种与保育工程实验室, 河北省微生物多样性研究与应用重点实验室, 河北保定 071002)摘要:复苏促进因子(Rpf)广泛存在于高 G+C 含量的革兰氏阳性细菌中,与休眠细胞的复苏有关,且不同细菌Rpf的种类和数量有一定差异.为了解不同物种中rpf基因的差别,对白色放线动孢菌(Actinokineospora alba)DSM 45114^T的3个Rpf蛋白的氨基酸序列、疏水性、信号肽、磷酸化位点、三级结构以及结构功能域进行生物信息学预测分析.设计了白色放线动孢菌(A. alba)DSM 45114^T rpf基因的特异性引物,从其基因组中扩增出了3个不同的rpf基因,随后构建pET-28a(+)-rpf表达载体,在Escherichia coli BL21(DE3)中进行诱导表达,成功表达了Rpf-2蛋白.将重组Rpf-2蛋白加入到活的非可培养(viable but non culturable,VBNC)状态的藤黄微球菌(Micrococcus luteus)和滨海红球菌(Rhodococcus marinonascens)中,结果表明,重组Rpf-2蛋白可以促进其复苏,添加量在体积分数为10%时复苏效果最好.本研究为后续探讨白色放线动孢菌(A. alba)Rpf蛋白的功能奠定了基础.关键词:白色放线动孢菌;复苏促进因子;异源表达;结构预测中图分类号:Q93 文献标志码:A 文章编号:1000-1565(2022)04-0411-10Cloning and expression of Actinokineospora alba DSM 45114^T rpf gene and structure prediction of Rpf proteinZHANG Yufan, LIU Jingjing, CAO Min, LIU Tongtong, CHEN Xue, ZHANG Xiumin(Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Engineering Laboratory of Microbial Breeding and Preservation of Hebei Province,Institute of Life Science and Green Development, School of Life Sciences, Hebei University, Baoding 071002, China)Abstract: Resuscitation-promoting factor(Rpf)is widely present in gram-positive bacteria with high G+C content and is related to the resuscitation of dormant cells. There are some differences in the types and quantities of Rpf among different bacteria. In order to understand the difference of rpf genes in different species, bioinformatics analysis was conducted on the amino acid sequence, hydrophobicity, signal peptide, phosphorylation site, tertiary structure and structural functional domain of three Rpf proteins in Actinokineospora alba DSM 45114^T. The specific primers for the three rpf genes of A. alba- 收稿日期:2022-01-03 基金项目:河北省重点研发计划项目(20326614D);河北省自然科学基金资助项目(C2020201005);河北大学2021年度研究生创新资助项目(HBU2021ss051) 第一作者:张宇帆(1997—),女,山西临汾人,河北大学在读硕士研究生,主要从事放线菌资源挖掘研究.E-mail:627256284@qq.com 通信作者:张秀敏(1970—),女,河北涞水人,河北大学教授,博士,主要从事微生物多样性及资源开发研究.E-mail:zhxiumin1106@126.com第4期张宇帆等:白色放线动孢菌 DSM 45114^T rpf基因的克隆表达及结构预测DSM 45114^T were designed, and corresponding genes were amplified. Subsequently, pET-28a(+)-rpf expression vectors were constructed and induced in Escherichia coli BL21(DE3), Rpf-2 protein was successfully expressed. Adding the recombinant Rpf-2 protein to the cells in viable but non culturable(VBNC)state of Micrococcus luteus and Rhodococcus marinonascens, the result showed that it could significantly promote their resuscitation, and the resuscitation effect was the most significant when 10% Rpf-2 protein was added. This laid a foundation for the subsequent study on the function of Rpf-2 protein in A. alba.

Key words: Actinokineospora alba, resuscitation-promoting factor(Rpf), heterologous expression, structure prediction

CLC Number:

ZHANG Yufan, LIU Jingjing, CAO Min, LIU Tongtong, CHEN Xue, ZHANG Xiumin. Cloning and expression of Actinokineospora alba DSM 45114^T rpf gene and structure prediction of Rpf protein[J]. Journal of Hebei University(Natural Science Edition), 2022, 42(4): 411-420.

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Cloning and expression of Actinokineospora alba DSM 45114^T rpf gene and structure prediction of Rpf protein

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