红麻MYB21基因的克隆、表达及载体构建  

Cloning,Expression Analysis and Vector Construction of MYB21 Gene in Kenaf

作　　者：钱景华[1] 周步进[1] 孔祥军[1] 李增强[1] 史奇奇 廖小芳[1] 周瑞阳[1] 陈鹏[1] 

机构地区：[1]广西大学农学院广西高校植物遗传育种重点实验室,南宁530004

出　　处：《生物技术通报》2016年第11期170-179,共10页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31260341;31560421);广西自然科学基金项目(2015GXNSFAA139059)

摘　　要：克隆红麻不育系和保持系的MYB21基因,分析MYB21基因在红麻不育系和保持系各器官的表达量,并构建过表达载体和RNAi载体,旨在为进一步研究该基因在红麻中的功能奠定基础。以同源克隆的方法克隆MYB21基因;用实时荧光定量的方法分析MYB21基因在红麻不育系和保持系不同组织器官的表达模式;根据酶切-连接的方法,构建过表达载体和RNAi载体。结果显示,红麻MYB21基因在不育系和保持中的基因序列没有差异,其c DNA全长序列为922 bp,包含843 bp的开放阅读框,DNA全长序列为1 108 bp,包含3个外显子和2个内含子(NCBI序列登录号为KT898146);MYB21基因主要在花药中表达,在不育系和保持系花药之间表达量呈极显著差异;成功构建MYB21过表达载体和RNAi载体。成功克隆MYB21基因的全长序列;实时荧光定量结果表明MYB21基因主要在花药中进行表达;构建的植物过表达载体PBI121-MYB21和RNAi载体p ART27-PK-R1-F2可用于该基因的功能研究。This work is to clone MYB21 gene and analyze its expression levels in different organs of kenaf for both cytoplasmic malesterility（CMS）and its maintainer,to construct over-expression vector and RNAi vector,and to lay the foundation for further study on thefunction of MYB21 gene in kenaf. The MYB21 gene was cloned using a homology method. The expression patterns of MYB21 in different organsfor CMS and its maintainer were analyzed by quantitative real-time PCR. Using enzyme digestion-ligation method,the expression vector andRNAi vector were constructed. As results,there was no difference on gene sequence between CMS and its maintainer. The full c DNA sequenceof MYB21 gene was 922 bp,including an 843 bp open reading frame. The full DNA sequence of MYB21 gene was 1 108 bp,containing 3 exonsand 2 introns（the NCBI Gen Bank accession number ：KT898146）. MYB21 gene was predominantly expressed in anther,the expression levelof MYB21 gene in anther between CMS and its maintainer was highly significant difference. Also the over-expression vector and RNAi vectorwere constructed successfully. In conclusion,the full-length sequence of MYB21 gene in kenaf is obtained,MYB21 gene is mainly expressedin anther of kenaf,and the over-expression vector PBI121-MYB21 and RNAi vector p ART27-PK-R1-F2 can be used for function study ofMYB21 gene.

关 键 词：红麻 细胞质雄性不育 MYB21基因 实时荧光定量 载体构建 

分 类 号：Q943.2[生物学—植物学] S563.5[农业科学—作物学]