娄素琳,林 鑫,黄思敏,等.莱茵衣藻CrDRBs基因的克隆和生物信息学分析[J].深圳大学学报理工版,2018,35(No.5(441-550)):523-528.[doi:10.3724/SP.J.1249.2018.05523]
LOU Sulin,LIN Xin,HUANG Simin,et al.Cloning and bioinformatics analysis of CrDRBs in Chlamydomonas reinhardtii[J].Journal of Shenzhen University Science and Engineering,2018,35(No.5(441-550)):523-528.[doi:10.3724/SP.J.1249.2018.05523]
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莱茵衣藻CrDRBs基因的克隆和生物信息学分析
娄素琳1,2,3,林 鑫1,3,黄思敏1,3,李 辉1,3,4,胡章立1,2,3,4

1)深圳大学生命与海洋科学学院,深圳市海洋生物资源与生态环境重点实验室,广东深圳 518060; 2)深圳大学光电工程学院,光电子器件与系统教育部/广东省重点实验室,广东深圳 518060; 3)广东省植物表观遗传学重点实验室,广东深圳 518060; 4)深圳大学龙华生物产业创新研究院, 深圳市海洋藻类生物工程技术研究中心,广东深圳 518060

分子生物学; 水生生物学; 莱茵衣藻; 微小RNA; 双链RNA结合蛋白; 基因克隆; 蛋白结构

Cloning and bioinformatics analysis of CrDRBs in Chlamydomonas reinhardtii
LOU Sulin1,2,3, LIN Xin1,3, HUANG Simin1,3, LI Hui1,3,4, and HU Zhangli1,2,3,4

1)College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Marine Bioresource & Eco-environmental Sciences, Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China2)College of Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China3)Guangdong Key Laboratory of Plant Epigenetics, Shenzhen 518060, Guangdong Province, P.R.China4)Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Longhua Innovation Institute for Biotechnology, Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China

molecular biology; hydrobiology; Chlamydomonas reinhardtii; micro ribonucleic acid(miRNA); double-stranded RNA-binding protein(DRB); gene cloning; protein structure

DOI: 10.3724/SP.J.1249.2018.05523

备注

双链核糖核酸(ribonucleic acid, RNA)结合蛋白(double-stranded RNA-binding protein, DRB)是一类含有双链RNA结合结构域(dsRBD)的蛋白,在小核糖核酸(micro ribonucleic acid, microRNA 或miRNA)的生物合成和作用方式选择方面起着重要作用.为获得莱茵衣藻(Chlamydomonas reinhardtii, C.reinhardtii)中所有CrDRBs的全长序列并进行生物信息学分析,从蛋白质功能结构域Pfam 数据库中下载了dsRBD结构域模块,使用 HMMER 软件基于 dsRBD检索C.reinhardtii全基因组序列,获得C.reinhardtii中4个含 dsRBD 结构域的 CrDRB 蛋白相关信息.抽提C.reinhardtii总RNA,反转录获得其 cDNA,设计相应引物克隆获得其完整的基因ORF序列.设计实时荧光定量PCR引物,以actin基因为内参,分析C.reinhardtii各CrDRBs基因的相对表达.同源比对分析C.reinhardtii各CrDRBs蛋白的dsRBD序列.通过Phyre2软件在线预测其dsRBD的高级结构.将莱茵衣藻CrDRBs的dsRBD与拟南芥的5个DRB成员构建系统进化树分析,初步探讨分析莱茵衣藻CrDRBs各成员的的进化保守性. 本研究共获得了4个莱茵衣藻双链RNA结合蛋白,均具有典型的dsRBD结构域,含有3个保守氨基酸.相比已报道的DUS16,其他3个CrDRBs的CDS序列较长,基因表达较高(除CrDRB2外).本研究为下一步揭示莱茵衣藻CrDRB在miRNA 调控途径的功能奠定基础.

Double-stranded RNA binding protein(DRB)is a class of proteins containing a double-stranded RNA binding domain(dsRBD), which plays a vital role in micro ribonucleic acid(microRNA/miRNA)biosynthesis and functions of miRNA. In order to obtain the homologues sequence of Chlamydomonas reinhardtii(C. reinhardtii)DRBs, the dsRBD domain module was downloaded from the protein functional domain database Pfam by bioinformatics technology, and the whole genome sequence of C. reinhardtii was retrieved by HMMER software. Four DRB homologues containing dsRBD domain, referred as DUS16、CrDRB1、CrDRB2 and CrDRB3, were successfully identified from the genome of C. reinhardtii by using Pfam and HMMER. These four homologues were next used to design specific primers, which led us to clone corresponding full length cDNA of CrDRBs genes in C. reinhardtii. The qRT-PCR result showed that the expression levels of CrDRB3 and CrDRB1 are significantly higher than those of DUS16 and CrDRB2. In addition, the results from homologous sequences alignment, in silico 3D protein structures and phylogenetic analysis suggested that CrDRBs are conservative in the evolution. In conclusion, we have successfully cloned four DRB homologues with typical dsRBD domains in green microalga C. reinhardtii. Compared with the reported DUS16, the other three CrDRBs have longer CDS sequences and higher gene expression. These results may lay the foundation for future research of revealing the functions of CrDRBs in the miRNA regulation pathway in green algae.

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