茶树EGCG合成过程相关的Ankyrin基因的克隆与表达分析

Molecular Cloning and Expression Analysis of Ankyrin, a Key Gene in the Process of EGCG Biosynthesis in Camellia sinensis L.

运用RACE技术,从茶树新品系"1005"嫩芽中克隆出Ankyrin基因全长c DNA(2 034 bp),5′UTR和3′UTR分别为353 bp和55 bp,编码541个氨基酸,命名为CS-Ankyrin。基因全长序列在线blast比对分析结果表明,该基因与葡萄、蓖麻、可可和杨树的Ankyrin序列的一致性分别为78%、78%、77%和77%。生物信息学分析显示,CS-Ankyrin锚蛋白重复序列是由5个ANK单元组成,该蛋白是定位在质膜上起作用的跨膜疏水蛋白,但疏水性较差;系统进化树分析表明,该基因编码的氨基酸与芝麻的亲缘关系最为接近。比较CS-Ankyrin在不同发育阶段叶片的表达和EGCG含量变化结果表明,CS-Ankyrin基因在3个品系不同样品的表达趋势与其EGCG含量变化趋势一致,芽头>二叶>四叶;亚细胞定位试验结果表明,CS-Ankyrin蛋白定位在细胞膜上起作用。推测该蛋白可能参与EGCG的储存和跨膜运输。

The full-length c DNA（2 034 bp） of one Ankyrin gene named CS-Ankyrin was cloned from the Camellia sinensis ＂1005＂ using RACE technique, which encoding a 541 amino acid protein. Bioinformatics analysis showed that,with low hydrophobicity, the encoded protein consisting of 5 ANK was hydrophobic and functioned in plasma membrane.Phylogenenetic analysis showed that the protein encoded by CS-Ankyrin had the closest genetic relationship with that in Sesamum indicum. Comparing the expression level of CS-Ankyrin and the EGCG content at different developmental stages of three cultivars, CS-Ankyrin expression was observed to vary with a similar trend observed for each cultivar：bud the second leaf the fourth leaf. The changes in EGCG was also found to follow a similar trend to that for gene expression. The subcellular localization results showed that the protein was located in the cytomembrane. We speculate that the protein is related to storage and transmembrane transport of EGCG.