华山姜中钙调素基因的克隆及其RNA原位杂交

Cloning and RNA in Situ Hybridization of Calmodulin Gene in Alpinia oblongifolia

本文报道了华山姜中钙调素cDNA的核苷酸序列以及由此推导的氨基酸序列,并用原位杂交的方法检测其在花器官中的时空表达模式.用[32P]d-CTP标记的小草蔻A lp in ia ha inanensis AGAM OUS(AG)cDNA的M ADS-dom a in作为探针筛选华山姜的cDNA文库,得到一个钙调素蛋白相关克隆,命名为A oCAM.华山姜钙调素A oCAM的cDNA全长518 bp,有一个包含149个氨基酸的开放读码框,编码区起始于第54个核苷酸,终止于第501个核苷酸.A oCAM与拟南芥、小麦、大豆、矮牵牛、玉米的钙调素氨基酸序列比较同源性高达95%.RNA原位杂交表明钙调素基因在花瓣、雄蕊、雌蕊细胞中大量表达.钙调素基因的表达强度随不同的发育阶段而变化:花发育早期在花的各器官中都表达强烈,以后逐渐减弱并向特定部位集中,如花粉囊、唇瓣、花柱和胚珠等分生能力较强的细胞中表达较强.

The paper reports nucleotide sequence and correspondingly deduced amino acid sequences of calmodulin in Alpinia oblongifolia as well as temporal and spatial expression models of calmodulin detected in Artemisia halodendron flowers by in situ hybridization. [^32p] d-CTP-labeled cDNA MADS-domain of Alpinia hainanensis AGAMOUS （AG） was used as the probe to screen the cDNA library of Alpinia oblongifolia and thus a clone relating with calmodulin protein was obtained and named AoCAM. The AoCAM cDNA of Alpinia oblongifolia calmodulin had a full length of 518 by and contained an open reading frame consisting of 149 amino acids whose coding region began with the fifty fourth nucleotide and ended with the five hundred and first nucleotide. The AoCAM had a homogeneity as high as 95% with the amino acid sequences of Arabidopsis thaliana CAM7,Triticum aestivum CAM, Glycine max CAM, Petunia hybrida CAM81 and Zea mays CAM2. The in situ hybridization revealed that calmodulin gene was expressed in large quantities in petal,stamen and pistil cells. The expression intensity of calmodulin gene varied with different developmental stages ,appearing high in the flower parts at the initial stage of flower development and then becoming low with strong expression focusing in specific locations such as pollen sac,labella,style ,ovule cells that had high dividing capabilities.