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

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。

Cloning and expression analysis of CtbZIP20 transcription factor gene from Carthamus tinctorius and construction of plant expression vector

Objective To clone bZIP20 (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 bZIP20 gene was cloned by RT-PCR techniques, and the protein characteristics were analyzed by bioinformatics, and phylogenetic tree was constructed. The expression of bZIP20 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR, and the plant expression vector pBASTA-bZIP20 was constructed. Results The ORF sequence of bZIP20 gene was 981 bp, encoded a protein of 326 amino acids (GenBank: KT692605). Sequence alignment and phylogenetic tree analyses showed that bZIP20 had 85.41% and 83.99% of consistency with bZIP of Sesamum indicum and Camellia assamica. Real-time PCR results showed significant differences, the highest expression level of bZIP20 gene was detected in flower, and was highest in the bud period, bZIP20 gene was significantly increased in root tissue inoculated with F. oxysporum. The plant expression vector pBASTA-bZIP20 was obtained. Conclusion The bZIP20 gene of safflower is successfully cloned, and the expression is analyzed. The plant expression vector pBASTA-bZIP20 is constructed.