潘那利番茄SpCDPK4基因的克隆、原核表达及表达模式分析

Cloning, prokaryotic expression and expression pattern analysis of SpCDPK4 gene in Solanum pennellii

基于同源克隆技术,本试验从野生潘那利番茄(Solanum pennellii)中获得一个钙依赖蛋白激酶家族基因CDPK4的全长序列;构建原核表达载体,并利用Western blot对重组蛋白进行验证;同时分析了该激酶的亚细胞定位情况及其在幼苗期不同组织的表达情况。结果表明,野生潘那利番茄SpCDPK4的开放阅读框长度为1 746 bp,编码581个氨基酸,理论蛋白分子量约为64.58 kDa,等电点为5.54,是非分泌型、亲水的稳定蛋白,且定位于细胞膜上。多序列比对及系统发育树分析表明, SpCDPK4编码的氨基酸序列与栽培番茄SlCDPK4 (XP_010327694.1)和马铃薯StCDPK4 (NM_001287877)相似度最高,相似性均为65.1%,与拟南芥AtCDPK20亲缘关系最近,归为第二类。SDS-PAGE电泳检测结果发现在70 kDa左右处有一条特异表达的蛋白条带,与预期目的产物大小一致,且Western-blot结果显示其能与anti-His单克隆抗体发生特异性反应。RT-qPCR结果表明, SpCDPK4在潘那利番茄不同组织中均有表达,且根中的表达量显著高于叶和茎;此外在盐胁迫下, SpCDPK4在根中的表达量迅速积累,且在1 h时达到峰值。

The full-length cDNA sequence of the CDPK4 from wild tomato Solanum pennellii was cloned by homology-based cloning. In the present study, we constructed the prokaryotic expression vector to induce the expression protein and then verified by Western blot. Moreover, a series of bioinformatics analysis, subcellular localization and expression pattern were performed. The results showed that the open reading frame length of SpCDPK4 was 1 746 bp, which encoded 581 amino acids and located in the cell membrane. The molecular weight of SpCDPK4 were 64.58 kDa and the isoelectric point was 5.54, which was a non-secretory and hydrophilic stable protein. Multiple sequence alignment and phylogenetic tree analysis showed that the amino acid sequence encoded by SpCDPK4 had the highest similarity with SlCDPK4(XP_010327694.1) and StCDPK4(NM_001287877). SDS-PAGE electrophoresis showed that there was a specific protein band at about 70 kDa, which was consistent with the expected size of the target product. Western blot showed that it could specifically react with anti his monoclonal antibody. RT-qPCR results showed that SpCDPK4 was expressed in different tissues of pannali tomato, and the expression level in root was significantly higher than that in leaf and stem. In addition, under salt stress, the expression level of SpCDPK4 in root accumulated rapidly, and reached the peak at 1 h.