大豆gma-miR1514b生物信息学分析及人工microRNA植物表达载体构建

Bioinformatic Analysis of gma-miR1514b and Construction of Its Artificial miRNA Plant Expression Vectors

MicroRNAs在植物发育与逆境响应等多个方面具有重要的调节作用。本研究采用生物信息学方法分析gmamiR1514b的靶基因和启动子中的顺式作用元件。PLACE和Plant CARE启动子在线预测工具分析表明gma-miR1514b启动子序列具有TATA-box、CAAT-box基本的顺式作用元件和一些参与光、逆境和植物激素应答相关的顺式作用元件。PMRD软件预测获得5个gma-miR1514b靶基因,分别与拟南芥CA2、NTL9和ANAC014基因同源。通过重叠PCR方法替换拟南芥At-MIR319的成熟链和互补链序列,构建含有amiR1514b前体的植物表达载体amiR1514b-p CAMBIA3301。

MicroRNAs play fundamental regulatory roles in various aspects of plant development and adaptive responses to stresses. In this study，the target gene and the promoter of gma-miR1514b gene were predicted and analyzed by bioinformatics methodology PMRD database analysis showed that precursor sequence length of gma-miR1514b was 109 bp, located in soybean 1 497 668-1 497 776［＋］of chromosome 17, and its mature sequence length was 21 bp. The predicted transcription start site of gma-miR1514b was found within 762 bp upstream of the precursor sequence.The database of PLACE and PlantCARE analysis showed that the promoter sequence contains basic elements TATA-box, CAAT-box and some cis-acting elements involved in response to light, stresses and plant hormones PMRD software analysis showed that 5 transcripts were targeted by gma-miR1514b, these targets were homologous with CA2, NTL9 and ANAC014 genes in Arabidopsis, respectively. The amiR1514b sequence was engineered into the miRNA and miRNA＊ of At-MIR319 using overlapping PCR and the amiR1514b backbone was transferred into the plant expression vector pCAMBIA3301.