龙眼胚性愈伤组织Cu/Zn-SOD分子伴侣基因CCS的克隆及其在体胚发生过程中的表达分析

为了解Cu/Zn超氧化物歧化酶分子伴侣基因(CCS)在龙眼体胚发生过程中的表达调控机制,以龙眼松散型胚性愈伤组织为材料,采用RT-PCR法,克隆了长960 bp含有完整开放阅读框的龙眼DlCCS核酸序列,其编码一个含有319个氨基酸的蛋白质(GenBank登录号:FJ973472).生物信息学分析显示:该蛋白为亲水的、稳定的、含有跨膜结构域的酸性蛋白质,定位于叶绿体;与毛果杨、葡萄、拟南芥、水稻和锦鸡儿具有较高同源性,含有植物CCS蛋白所特有的3个保守结构域;预测其通过不同的磷酸化方式,改变酶活性及蛋白构象,参与龙眼体胚中超氧化物代谢以及金属离子转运等生物学过程,在氧化还原过程中发挥作用.利用实时荧光定量PCR(qPCR)技术研究该基因在龙眼体胚发生发育过程中的表达情况,结果显示,从松散型胚性愈伤组织(Stage 1)到胚性紧实球形结构阶段(Stage 4),DlCCSmRNA的转录水平较低且波动小;当胚性紧实球形结构进一步发育到子叶形胚阶段(Stage 8),其表达量迅速增加,在成熟胚阶段(Stage 9)达到最高峰,提示DlCCS可能在龙眼体胚的中晚期起主要作用

Cloning of Copper Chaperone for Superoxide Dismutase Gene CCS from Embryogenic Callus of Dimocarpus longan Lour. and Its Expression Analysis During Somatic Embryogenesis

Copper chaperone for superoxide dismutase gene(CCS) was cloned and its expression level was analyzed for elucidating its role during longan somatic embryogenesis.DlCCS gene was isolated by RT-PCR,containing a 960-nucleotideslong open reading frame(ORF) which encoded a protein of 319 amino acid residues.Bioinformatics analysis showed that the protein encoded by DlCCS with the signal peptide and transmembrane helices was hydrophilic,stable and acidic protein,located in the chloroplast.Homology researches with the deduced amino acid residues indicated that DlCCS gene had a high similarity to other plant CCS genes,and the predicted DlCCS polypeptide contained 3 distinct domains.The phosphorylation occurred mainly on serine,followed by threonine and thyosine residues.The DlCCS protein was involved in superoxide metabolism and metal ion transport process,and played a role of oxidation-reduction during longan somatic embryogenesis.qPCR results indicated that DlCCS was expressed at low levels from the friable-embryogenic callus(Stage 1) to the compact pro-embryogenic cultures(Stage 4).With further embroygenic development,its expression increased rapidly and peaked at the mature embryo stage(Stage 9),suggesting that DlCCS might play a major role in the middle and late developmental stages of longan somatic embryogenesis.