钙调蛋白(CaM)是生物细胞内的重要调控蛋白,参与调节细胞内一系列酶的活性及各项生命活动。应用RACE和RT-PCR技术克隆了白桦的钙调蛋白基因CaM的全长cDNA,该基因长631 bp,开放阅读框(ORF)为450 bp,编码149个氨基酸,预测编码蛋白的相对分子质量为16.85 kDa,理论等电点为4.11,经过对其推测的氨基酸序列进行功能结构域预测,具备CaM典型的EFh结构域,将该基因命名为BpCaM。多序列比对与系统进化树分析结果显示,该基因序列同其它植物的CaM序列相似性均在97%以上。以白桦actin基因作为内参基因,利用定量PCR技术分析BpCaM基因在各发育时期的表达情况,结果表明,BpCaM基因在营养器官的表达高于花器官,幼嫩的组织要高于成熟组织,白桦BpCaM基因在各个器官组织均有表达,但表达量有差异。另外,对白桦雄花突变体进行了转录表达分析,结果显示在雌花、幼茎和幼叶中BpCaM基因在突变植株中上调表达,说明BpCaM基因参与多种组织的发育。
Calmodulin(CaM) is a key regulating protein in cells,participating in and regulating enzyme activities and life activities.Applying RACE and RT-PCR methods,BpCaM gene was cloned from Betula platyphylla.The BpCaM gene with 631 bp in length contains 450 bp open reading frame and encodes a 149-amino-acid polypeptide with a predicted relative molecular mass of 16.85 kDa and pI of 4.11.The functional domains of its deduced amino acid sequence were confirmed as typical EFh motifs of CaM.The isolated gene was named as BpCaM.The results of multiple sequences alignment and the phylogenetic tree analysis showed that it shared more than 97% identity with other plant species.Expression of BpCaM was analyzed by the method of quantitative PCR normolized by actin gene in B.platyphylla.The results showed that BpCaM was more highly expressed in vegetative tissues than in reproductive tissues,and more higly in young tissues than in aging tissues.BpCaM expressed in every tissues at different levels.Furthermore,transcription and expression of BpCaM was analyzed in a natural male inflorescense mutant,the results showed that BpCaM up-expressed was detected in female inflorscences,young shoot and young leaves,indicating that it was associated with development of many tissues.