莱茵衣藻MAPK4基因RNAi载体的构建及鉴定
Construction and Identification of MAPK4 Gene RNAi Vector of Chlamydomonas reinhardtii
王子贺 1邓晓东 2张阳 1费小雯1
作者信息
- 1. 海南医学院基础医学与生命科学学院,海南海口 571101
- 2. 中国热带农业科学院热带生物技术研究所,海南海口 571101
- 折叠
摘要
[目的] 构建莱茵衣藻2A38的MAPK4基因的RNAi载体. [方法]提取莱茵衣藻细胞总RNA,利用PCR扩增出编码MAPK4的基因片段,用基因工程技术将MAPK4基因片段连接载体pMD18-T上,经过2次不同的双酶切后,与RNAi中间载体pT282连接,最后连接到干涉载体pMaa7IR/XIR上,用酶切及测序鉴定MAPK4基因RNAi载体并转化莱茵衣藻. [结果]经限制性内切酶酶切及测序鉴定,证实为重组质粒,并且该重组载体可在莱茵衣藻中表达. [结论]构建的MAPK4基因RNAi载体结构正确,为进一步利用RNAi技术使MAPK4基因沉默表达,研究MAPK4在莱茵衣藻中的生物学功能奠定了试验基础.
Abstract
[Objective]To construct a MAPK4 gene RNAi vector of Chlamydomonas reinhardtii 2A38. [Method]Total RNA was extracted from C. reinhardtii, and the MAPK4 gene was amplified by reverse transcription polymerase chain reaction(PCR). By using gene engineering technique, C. reinhardtii MAPK4 gene was inserted to pMD18-T vector. The forward and reverse fragments ,which were obtained by two different double enzyme digestion of pMD18-T vector, were inserted into the RNAi intermediate vector pT282. The inverted-repeat fragment was digested by enzyme EcoRI and linked to the vector pMaa7IR/XIR.The recombined plasmid was identified by digestion and sequencing. [Result]Through restriction enzyme digestion and nucleotide sequencing identification, the recombinant vector was successfully constructed, it could be expressed in C. reinhardtii. [Conclusion]The constructed MAPK4 gene RNAi vector has correct structure, which laies the foundation for using RNAi technique to silence the gene expression and further research on MAPK4 biological function in C. reinhardtii.
关键词
莱茵衣藻/MAPK4基因/RNAiKey words
Chlamydomonas reinhardtii/MAPK4 gene/RNAi引用本文复制引用
出版年
2017
国家科技期刊平台
NSTL
维普
万方数据