红花bZIP20转录因子基因的克隆、表达分析及植物表达载体构建

Cloning and expression analysis of Ctb ZIP20 transcription factor gene from Carthamus tinctorius and construction of plant expression vector

目的克隆红花花瓣中b ZIP20(Basic region/leucine zipper motif)基因,研究其在不同组织中的表达量并构建其植物表达载体。方法根据红花转录组测序结果挑选b ZIP基因的设计引物,以红花花瓣总RNA为模板,采用RT-PCR法扩增b ZIP20基因开放阅读框(ORF)片段,利用RT-PCR法分析在红花不同组织以及尖孢镰刀菌侵染后红花根部b ZIP20基因的表达量,同时构建植物表达载体p BASTA-b ZIP20。结果 b ZIP20基因ORF长981 bp,编码326个氨基酸(Gen Bank登录号为KT692605)。红花b ZIP20与其他物种氨基酸具有一定的同源性,其与芝麻、野茶树的氨基酸序列相似性高达85.41%和83.99%。实时荧光定量PCR分析表明,b ZIP20基因在不同组织中的表达水平具有显著差异,在花中呈现高表达,而在其他组织中低表达。接种尖孢镰刀菌的红花根部组织中b ZIP20基因的表达显著上调。结论成功地对b ZIP20基因进行克隆及表达分析,并构建植物表达载体p BASTA-b ZIP20。

Objective To clone b ZIP20（basic region/leucine zipper motif） gene from Carthamus tinctorius, analyze the expression level in different plant tissues, and construct the plant expression vector. Methods The b ZIP20 gene was cloned by RT-PCR techniques, and the protein characteristics were analyzed by bioinformatics, and phylogenetic tree was constructed. The expression of b ZIP20 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR, and the plant expression vector p BASTA-b ZIP20 was constructed. Results The ORF sequence of b ZIP20 gene was 981 bp, encoded a protein of 326 amino acids（Gen Bank： KT692605）. Sequence alignment and phylogenetic tree analyses showed that b ZIP20 had 85.41% and 83.99% of consistency with b ZIP of Sesamum indicum and Camellia assamica. Real-time PCR results showed significant differences, the highest expression level of b ZIP20 gene was detected in flower, and was highest in the bud period, b ZIP20 gene was significantly increased in root tissue inoculated with F. oxysporum. The plant expression vector p BASTA-b ZIP20 was obtained. Conclusion The b ZIP20 gene of safflower is successfully cloned, and the expression is analyzed. The plant expression vector p BASTA-b ZIP20 is constructed.