中华绒螯蟹Smad3(EsSmad3)的cDNA克隆、序列分析及表达特征  

Molecular cloning, sequence analysis, and tissue expression of Smad3-like protein from Eriocheir sinensis

作　　者：田志环[1,2] 焦传珍[1] 成永旭[2] TIAN Zhihuan;JIAO Chuanzhen;CHENG Yongxu(College of Yingdong Life Science,Shaoguan University,Shaoguan 512005,China;Key Laboratory of Freshwater Fishery Germplasm Resources,Ministry of Agriculture;Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]广东韶关学院英东生命科学学院,广东韶关512005 [2]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306

出　　处：《中国水产科学》2018年第2期316-324,共9页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(31572635);科技部港澳台科技合作专项项目(2014DFT30270);上海市科学技术委员会科研项目(16DZ2281200);上海高校水产学高峰学科建设项目(2015-62-0908);上海市科技兴农推广项目[沪农科推字(2015)第1-7号];韶关学院生态学重点扶持学科(230079030101)

摘　　要：为了研究Smad基因在中华绒螯蟹(Eriocheir sinensis)蜕壳前后肌肉生长过程中的功能,应用RACE技术克隆得到编码中华绒螯蟹Smad3(命名为Es Smad3)的cDNA全长序列2021 bp,包括36 bp的5′非翻译区(5′-UTR)、656 bp的3′非翻译区(3′-UTR)和编码442个氨基酸的开放阅读框。蛋白质结构域分析显示EsSmad3含有MH1和MH2两个特征性保守结构域。多序列比对显示,EsSmad3与人、斑马鱼、黑腹果蝇中的同源蛋白序列一致性分别为0.679、0.691、0.619。应用荧光定量RT-PCR技术分析EsSmad3在性成熟中华绒螯蟹各组织及幼体不同蜕壳时期不同部位肌肉组织中转录水平上表达量的变化。结果显示,Es Smad3在性成熟个体的肝胰腺、眼柄、表皮、卵巢、精巢、心脏、螯足、鳃、三角膜等组织中均有表达,其中眼柄和精巢中表达量较高,心脏和肝胰腺中表达量最低。在幼体不同蜕壳时期的不同部位的肌肉中,Es Smad3表达量变化不同:步行足肌肉组织中EsSmad3 mRNA表达在蜕壳间期高于蜕壳前D_(3–4)期和蜕壳后A^B期,但无显著的统计学差异(P>0.05)。螯足肌肉在蜕壳前晚期D_(3–4)期急剧下调(P<0.05),蜕壳后A^B期开始表达量显著升高(P<0.05),直至蜕皮间期C期。腹部肌肉组织中EsSmad3m RNA水平在蜕皮间期C期显著高于蜕壳后A^B期,这种上调一直持续到蜕壳前晚前期D_(3–4)。上述结果表明,Es Smad3在中华绒螯蟹蜕壳过程中不同部位肌肉组织中的表达模式与蜕皮周期密切相关,推测Es Smad3参与了中华绒螯蟹蜕壳诱导的肌肉萎缩、生长及重建过程。In the present study,full length cDNA,encoding the Smad3 from Eriocheir sinensis(EsSmad3),was cloned using 3′Race and 5′Race techniques,and the sequence and structural analysis of EsSmad3 was conducted using bioinformatics methods.The results showed that the full-length cDNA encoding EsSmad3 consisted of 2021 bp nucleic acids in length,including a 5′-UTR of 36 bp,a 3′-UTR of 656 bp and an open reading frame(ORF)which encoded 442 amino acids.Analysis of the protein domain features showed that the deduced polypeptides contained two conservative domains characteristic of MH1 and MH2.Multiple sequence alignment revealed that the amino acid sequences of EsSmad3 have the 0.679,0.691,and 0.619 identity with Homo sapiens,Brachydanio rerio,and Drosophila melanogaster respectively.The tissue distribution of EsSmad3 mRNA in sexually mature individuals and different muscle groups during the molt cycle in juvenile crabs,were analyzed using quantitative real-time PCR(qRT-PCR).In sexually mature crabs,the EsSmad3 transcript was detected in the eyestalk,claw muscle,ovary,heart,hepatopancreas,epidermis,testicle,gill,and triangular membrane,and the expression level was relatively high in the eyestalk and testicle,and was low in the hepatopancreas and heart.In juvenile crabs,the EsSmad3 transcript in different muscle groups was different depending on the molt stage.In walking leg muscles,the EsSmad3 expression level was higher in inter-molt C stage than in the later pre-molt D3-4 and post-molt A-B stages,but there was no statistically significant difference(P>0.05).In claw muscles,the EsSmad3 expression level decreased rapidly in the pre-molt D3–4 stage(P<0.05)and increased in the post-molt A-B stage,lasting to the inter-molt C stage(P<0.05).In abdominal muscles,the EsSmad3 expression level was much higher in the inter-molt C stage than in the post-molt A-B stage(P<0.05),and this up-regulation continued to the pre-molt D3-4 stage.These results suggest that the expression of EsSmad3 transcript in different muscle groups wa

关 键 词：SMAD3基因 基因克隆 肌肉生长 蜕壳 中华绒螯蟹 

分 类 号：S917[农业科学—水产科学]