合浦珠母贝C-型凝集素基因的序列特征和功能分析  

Sequence features and functional analysis of the C-type lectin gene(PoLEC1)from pearl oyster Pinctada fucata

作　　者：胡钰婷[1,2] 张殿昌[2] 崔淑歌[2,3] 郭华阳[1,2] 陈明强[2] 江世贵[2] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院南海水产研究所,广东广州510300 [3]暨南大学生命科学技术学院,广东广州510632

出　　处：《水产学报》2011年第9期1327-1336,共10页Journal of Fisheries of China

基　　金：中央级公益性科研院所基础科研业务费专项资金项目(2009TS23)

摘　　要：通过EST筛选结合重测序法获得合浦珠母贝一种C-型凝集素cDNA的全长序列(命名为PoLEC1)。PoLEC1全长988 bp,5'-UTR为33 bp,3'-UTR为101 bp,开放阅读框为864 bp,编码287个氨基酸,分子量为31.68 ku,理论等电点约5.83。预测的氨基酸序列中含有信号肽(Met1-Ser19)和糖结合位点。同源性分析结果表明,PoLEC1与其它物种C-型凝集素的的糖识别结构域序列的同源性在18.8%～28.6%,相似性在28.9%～50.0%之间。进化树分析结果表明,PoLEC1与栉孔扇贝聚为一支。组织表达分析表明,PoLEC1 mRNA在消化腺、外套膜、性腺、闭壳肌、肠、鳃和血淋巴组织均有表达,在消化腺中的表达分析表明,经溶藻弧菌刺激后2 h表达显著下调,而在4和24 h后表达显著上调。利用PoLEC1成熟肽构建原核表达载体,在大肠杆菌中进行重组表达。制备包涵体后,用IDA HisBind树脂纯化得到单一的重组蛋白,凝菌试验表明该蛋白对大肠杆菌有明显的凝集作用。Pinctada fucata is one of the main shellfishes which produce sea water pearls,and it has high economic value.Shellfish diseases continuing to occur in recent years,we had to strengthen to study the immune system of shellfish.In this test,through researching the related genes C-type lectin from P.fucata,provided some basic theories of molecular assisted selection for P.fucata.We identified and cloned the cDNA of the C-type lectin gene（PoLEC1）from P.fucata by the cDNA library of P.fucata.Sequence analysis showed that PoLEC1 is 998 bp long,a 5′-UTR is 33 bp,and a 3′-UTR is 101 bp,open reading frame is 864 bp,encoding 287 amino acids,the molecular weight is 31.68 ku and the theoretical isoelectric point is about 5.83.The signal peptide in the predicted amino acid is the Met1-Ser19,also contains sugar binding sites.Homology analysis showed that the homology of PoLEC1 and other species.Amino acid sequence is between 18.8% and 28.6%,the similarity is between 28.9% and 50.0%.The phylogenetic analysis showed that the PoLEC1 shared the same branch with Chlamys farreri.Tissue expression analysis showed that the PoLEC1 mRNA was expressed in digestive gland,mantle,gonad,adductor muscle,intestine,gills and hemolymphe.Digestive gland expression analysis showed that after stimulation by Vibrio alginolyticusit was significantly reduced in 2 h,and expression was up-regulated in 4 h and 24 h.The prokaryotic expression vector of PoLEC1 was constructed using the mature peptide,and expressed in E.coli.After the preparation of inclusion bodies,purified by IDA HisBind resin and a single protein.And the coagulation test showed that the protein can agglutinate the E.coli significantly.

关 键 词：合浦珠母贝 C-型凝集素基因 RT-PCR 原核表达 

分 类 号：Q753[生物学—分子生物学] S917.4[农业科学—水产科学]