栉孔扇贝BI-1基因的克隆与表达分析

本研究采用RT-PCR和RACE技术首次获得了栉孔扇贝(Chlamys farreri)BI-1基因(Cf BI-1)c DNA全长序列,长度957bp,5′和3′非编码区长度分别为48bp和195bp,开放阅读框为714bp,预测编码一个含有237个氨基酸的蛋白质,分子量为27k Da;结构预测显示,Cf BI-1蛋白包含6个跨膜结构域。同源和分子进化聚类分析显示,Cf BI-1蛋白序列与其他一些物种中的BI-1蛋白序列相似性很高,表明BI-1具有很高的保守性。通过荧光定量PCR的方法检测了Cf BI-1 m RNA在栉孔扇贝正常组织中和在干露、脂多糖以及扇贝急性病毒性坏死症病毒刺激之后的表达水平的变化。结果表明,Cf BI-1在栉孔扇贝各组织中广泛分布,其中闭壳肌中的表达量最高,血淋巴中的表达量最低。在干露和病毒刺激以后,Cf BI-1基因表达水平较对照组都有显著上调(P0.05),表明Cf BI-1基因可能参与了干露刺激和急性病毒性坏死症病毒感染后的细胞应激响应及其诱导的细胞凋亡过程。综合上述分析,我们认为Cf BI-1基因在栉孔扇贝细胞凋亡调控过程中可能起到重要作用,可为栉孔扇贝凋亡相关基础研究提供参考。

CLONING AND EXPRESSION OF BAX INHIBITOR-1 GENE OF ZHIKONG SCALLOP CHLAMYS FARRERI

The full length cDNA of Bax inhibitor-1 in Zhikong scallop Chlamys farreri (CfBI-1) was first cloned using RT-PCR and rapid amplification of cDNA ends (RACE). A 957-bp sequence was acquired. The sequence contains a 48-bp 5' untranslated region (5'-UTR), a 195-bp 3'-UTR, and an open reading frame (ORF) of 714bp encoding a polypeptide of 273 amino acid in molecular weight of 27kDa. The homology and phylogenetic analysis revealed that the amino acid sequence of CfBI-1 showed high homology with that of other species, indicating that CfBI-1 was highly conservative. We used quantitative real-time PCR to estimate mRNA expression level of CfBI-1 in various tissues and its induced expression following air exposure stimulation, lipopolysaccharide infection, and scallop acute viral necrobiotic disease virus (AVNV) infection. The results show that CfBI-1 expression existed in all tested tissues of Chlamys farreri, including adductor muscle, digestive gland, gill, mantle, gonad, and haemolymph. CfBI-1 expression was the highest in adductor muscle and lowest in haemolymph. The levels of CfBI-1 mRNA increased clearly after air exposure stress and AVNV infection, implying that CfBI-1 may be involved in the apoptosis process induced by air exposure stimulation and AVNV infection. Therefore, CfBI-1 may play an important role in apoptosis of Chlamys farreri, which will provide new data for apoptosis study of the scallop.