木榄CaM基因的克隆及序列分析

The Cloning and Research of CaM on Bruguiera gymnorrhiza

以深圳红树自然保护区的木榄为材料,根据GenBank上已公布的钙调蛋白氨基酸保守区的碱基序列设计引物,RT-PCR扩增木榄CaM序列,获得cDNA全长735bp,命名为BgCaM(GenBank登录号:GU722140)。生物信息学分析表明,BgCaM与拟南芥、花生、大麦、榉木及甘薯等植物钙调蛋白氨基酸保守区具有较高同源性。BgCaM基因完整开放阅读框为450bp,编码149个氨基酸,预测蛋白等电点为4.11,分子量为16.8kD。分析基因序列和相应酶切位点,将扩增获得的BgCaM基因完整开放阅读框与中间表达载体相应酶切产物进行连接,构建植物表达载体pC13rd29A-BgCaM,为BgCaM功能分析和进一步利用奠定了基础,为植物转基因研究与应用又增添了一个新的基因源。

Bruguiera gymnorrhiza as materials in this study which collected from the Shenzhen mangrove nature reserves.This study referenced other CaM genes have been reported from GenBank.Based on the highly conserved nucleotide sequence in the region of amino acids,we cloned the full length CaM cDNA sequence which is 735 bp from Bruguiera gymnorrhiza,and named BgCaM（GenBank number：GU722140）.Bioinformatics analysis showed that the BgCaM has high homology on conservative region of amino acids when compared with the Arabidopsis thaliana,peanuts,barley,beech,sweet potato and other plants by sequence alignment and phylogenetic tree analysis.The complete open reading frame of BgCaM is 450 bp,encoding 149 amino acids.Predicted protein isoelectric point is 4.11,molecular weight is 16.8 kD.Analysis of gene sequences and the corresponding restriction sites,Digest the PCR amplification product including the complete reading frame and introduce the fragment into the digested intermediate expression vector,and construct plant expression vector pC13rd29A-BgCaM.This study has laid a foundation to make function analysis and application on BgCaM,it provide a new genetic sources for plant transgenic research and application.