采用同源克隆策略和RACE技术,从三角帆蚌(Hyriopsis cumingii)外套膜组织中成功克隆得到钙网蛋白(Calreticulin, CRT)基因的全长cDNA序列,共1838 bp,开放阅读框为1257 bp,编码418个氨基酸,5′端非编码区为75 bp,3′端非编码区为506 bp,基因序列提交GenBank的登录号为JX416227。生物信息学分析表明,三角帆蚌钙网蛋白基因具有一段信号肽序列、两条典型的钙网蛋白家族标签序列 KHEQNIDCGGGY和IMFGPDICG、三个保守的 N-、P-和 C-端功能域及内质网前导序列 HDEL。NJ 法系统进化分析显示三角帆蚌首先与海洋双壳类紧密聚在一起,且与蚯蚓等环节动物亲缘关系较近,聚为一支,然后依次与虾类、昆虫、鱼类、两栖类、哺乳类聚在一起。经荧光定量 PCR 检测,钙网蛋白基因在三角帆蚌的外套膜、闭壳肌、斧足、鳃、肝脏、性腺、心脏、肠等8个组织中均有表达,其中在外套膜、鳃和斧足等与贝类钙代谢相关的组织中表达量较高预示其可能参与三角帆蚌的钙代谢。不同 Ca2+浓度处理试验的结果表明,随着水体中 Ca2+浓度逐渐升高,三角帆蚌钙网蛋白基因在外套膜中的表达水平呈先上升后下降的趋势,并在60 mg/L 时达到最高峰,表明适宜的 Ca2+浓度可促进钙网蛋白基因表达,而过高的 Ca2+浓度则会抑制其表达。同时在60 mg/L Ca2+浓度条件下,对三角帆蚌外套膜进行不同时间的表达试验,结果表明钙网蛋白基因的表达量随时间推移先上升,并于48h达到最大表达量,而后逐渐下降。上述结果为进一步深入研究钙网蛋白基因的功能及其调控机理奠定基础。%The full-length cDNA sequence of Calreticulin CRT gene was isolated from the mantle of Hyriopsis cumingii by using homology cloning strategy and SMART RACE technique. The entire CRT cDNA was 1838 bp, containing a 1257 bp complete open reading frame which encoding a protein with 418 amino acids, a 75 bp 5′UTR, and a 506 bp 3′UTR (GenBank accession number is JX416227). Bioinformatics analysis indicated that the calreticulin gene had a sig-nal peptide, two typical calreticulin family labels (KHEQNIDCGGGY and MFGPDICG), three conserved domains (N-, P-, and C-), and the endoplasmic reticulum retrieval sequence HDEL. Phylogenetic analysis indicated that the CRT gene of H. cumingii was closely related to seawater bivalves, followed by that of annelidas and then fish, amphibians, and mammals. Real-time PCR revealed that CRT gene was ubiquitously expressed in all tested tissues, but far more abun-dant in tissues that closely relating to calcium metabolism such as mantle, gill and foot. This result indicates an intrin-sical relationship between CRT gene and calcium metabolism in H. cumingii. When exposed to a serie of increasing Ca2+, the mRNA expression of CRT gene in mantle was shown to be bell-shaped, ascending when Ca2+concentration was less than 60 mg/L and descending when Ca2+concentration was greater than 60 mg/L. The result that CRT gene expression reached its maxium when exposed to 60 mg/L Ca2+, suggesting appropriate Ca2+concentration would stimulate the ex-pression of CRT gene. Moreover, as the Ca2+(60 mg/L) exposure time increased, the CRT gene expression in mantle was shown to increase initially, reach its peak at 48h, and then decrease subsequently. The results of present study will provid useful information for further studies on function and regulation mechanism of CRT gene.
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