[1]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(5):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(5):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()
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《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第35卷
期数:
2018年第5期
页码:
523-528
栏目:
生物工程
出版日期:
2018-09-25

文章信息/Info

Title:
Cloning and bioinformatics analysis of CrDRBs in Chlamydomonas reinhardtii
作者:
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
Author(s):
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.China 2) 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.China 3) Guangdong Key Laboratory of Plant Epigenetics, Shenzhen 518060, Guangdong Province, P.R.China 4) Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Longhua Innovation Institute for Biotechnology, Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China
关键词:
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
Keywords:
molecular biology;  hydrobiology;  Chlamydomonas reinhardtii;  micro ribonucleic acid (miRNA);  double-stranded RNA-binding protein (DRB);  gene cloning;  protein structure
分类号:
Q 756
DOI:
10.3724/SP.J.1249.2018.05523
文献标志码:
A
摘要:
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，其它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．
Abstract:
Double-stranded ribonucleic acid binding proteins (DRB), containing a double-stranded RNA binding domain (dsRBD), plays vital roles in micro ribonucleic acid (microRNA / miRNA) biosynthesis and involve in determining the action mode of miRNAs regulation. In order to acquire the full length of all the CrDRBs genes in Chlamydomonas reinhardtii (C. reinhardtii) and bioinformatics analysis of them, dsRBD sequence is downloaded from the database of Pfam. Four CrDRB genes which contain dsRBD domain are found in C. reinhardtii, based on the soft HMMER to index the whole genome sequence based on sequence of dsRBD. Extracted total RNA from C. reinhardtii, gained their cDNAs according to reverse transcription and designed the corresponding primers to clone and acquire the full length of CrDRBs genes. Primers of quantitative real-time PCR are designed to analyze the relative expression of CrDRBs genes in C. reinhardtii, using Actin gene as the internal reference. Analyzed the dsRBD homologies from all the CrDRB members in C. reinhardtii. 3D structure of Chlamydomonas dsRBD is predicted using the online phyre2 software. The evolution tree of the dsRBDs from Chlamydomonas and five DRB members of Arabidopsis is built to preliminarily discuss the evolutionary conservation of the CrDRB members. In this study, four CrDRBs which have a typical dsRBD domain, and with three conserved amino acids were gained from C. reinhardtii. Compared with reported DUS16, the CDS sequences of the other three CrDRBs are longer, and the gene expression is also higher (except CrDRB2). All of these lay the foundation for future research of revealing the function of CrDRBs in the miRNA regulation pathway.

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更新日期/Last Update: 2018-08-21