大豆gma-miR1508a靶基因鉴定及植物表达载体构建

Validation of Selected gma-miR1508a Targets and Construction of Its Plant Expression Vectors

MicroRNAs是内源的非编码小RNA分子,在植物生长、发育和逆境响应过程中具有重要的调控功能。本研究采用5'RLM-RACE方法鉴定了大豆gma-miR1508a的1个靶基因Glyma16g27800。为了构建gma-miR1508a的过表达载体,使用烟草花叶病毒组成型启动子35S控制gma-miR1508a的过表达,采用PCR方法从大豆基因组DNA中扩增了101 bp含有折回结构的前体序列,扩增片段被亚克隆至p CAMBIA3301载体中,PCR和测序结果表明gma-miR1508a植物表达载体构建成功。

MicroRNAs（ miRNAs） are endogenous,noncoding,small RNAs that have essential regulatory functions in plant growth,development,and stress response processes. In this study,one target gene of gma-miR1508 a,Glyma16g27800,was verified using a modified 5＇ RLM-RACE（ RNA ligasemediated rapid amplification of 5＇ c DNA ends） assay. To generate an overexpression construct that constitutively overexpressed gma-miR1508 a under the control of a cauliflower mosaic virus 35 S promoter,a 101 bp fragment flanking the miRNA sequence including the fold-back structure was amplified from soybean genomic DNA by PCR. The amplified fragment was subcloned into the p CAMBIA3301 vector. PCR and sequencing results suggest that plant expression vector of gma-miR1508 a gene was constructed successfully. This constructed vectors provided an effective too1 for the further study of gma-miR1508 a gene function.