油茶钙调素基因的cDNA克隆与表达分析

Cloning and expression analysis of calmodulin cDNA genes from Camellia oleifera

在构建油茶近成熟种子cDNA文库的基础上,分离鉴定了油茶钙调素基因CoCaM1、CoCaM2的cDNA序列(Gen Bank登录号:EU856536和FJ649316),它们的长度分别为953 bp和1024 bp,均含有447 bp的开放读码框,编码的149个氨基酸完全相同;它们编码区的核苷酸序列高度一致,仅有25个碱基替代,证实了"多个基因编码一种蛋白"的假设。该蛋白含有19种氨基酸,属于酸性亲水性蛋白,理论等电点4.10,相对分子量16.83 kDa;它含有EF-手臂、半胱氨酸等结构域。该蛋白的亲水性/疏水性、柔性、抗原性具有较好的一致性,并表现高度的柔性。Blast及遗传进化分析表明:该蛋白的氨基酸序列与其它植物钙调蛋白的氨酸序列同源性较高。油茶CoCaM1在花芽到子房形成中的表达量较高;而CoCaM2在果实形成、膨大、油脂积累过程中的表达量较多,而在叶片和成熟种子中较少。这表明它们在花芽发育、果实形成、种子油脂合成积累中发挥着不同的调控作用。

Based on the conducted cDNA library of Camellia oleifera, two full-length cDNA genes were obtained, and were 953 bp and 1 024 bp, respectively, designated as CoCaM1 and CoCaM2（Gen Bank access numbers： EU856536 and FJ649316）. They shared high similarity at nucleotide level in the coding regions（CDS）. They both contained the 447-bp CDS with 25 nucleotide substitutions, and encoded an identical polypeptide of 149 amino acids（predicted relative molecular weight of 16.83 k Da）. The protein is a hydrophilic acidic protein, and comprised of 19 amino acids with p I（theoretical isoelectric point） of 4.10. The characteristic is consistent with the hypothesis ＂Multigenes possess an identical amino acid sequence＂. It possesses four EF-hand domains, cysteine and other enzymebinding sites, etc. The protein has some identical degree in hydrophilicity, flexible regions and antigenicity, and thus shows high flexibility. The blast and phylogenetic analysis indicated that the amino acid sequence of C. oleifera Ca M displayed highly homologous compared with those of other higher plants. The higher expression levels of CoCaM1 were observed in the stage from flower bud to ovary formation. The expression levels of CoCaM2 were much higher in fruit formation and oil accumulation, while in leaves and mature seeds were lower. This shows that they may play different roles in the development of flower buds, fruit formation and oil synthesis and accumulation.