中华绒螯蟹Akt基因的cDNA克隆、序列分析及表达特征  

Molecular cloning,sequence analysis and tissue expression of serine/threonine kinases Akt from Eriocheir sinensis

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

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：《水产学报》2018年第4期485-494,共10页Journal of Fisheries of China

基　　金：国家自然科学基金(31572635);科技部港澳台科技合作专项(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：为研究Akt基因在中华绒螯蟹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Akt(命名为Es Akt)的全长为2 200 bp的c DNA序列,包括159 bp的5′非翻译区(5′-UTR)、496 bp的3′非翻译区(3′-UTR)和长度为1545 bp编码514个氨基酸的编码序列。蛋白质结构域分析显示Es Akt含有丝氨酸/苏氨酸蛋白激酶家族的3个特征性保守结构域。多序列比对和系统发育分析显示,Es Akt与中国明对虾、凡纳滨对虾的Akt序列一致性都为0.889,在系统发育树中节肢动物Akt形成一个分支。应用荧光定量RTPCR技术分析Es Akt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 Akt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,其中卵巢、眼柄和精巢中表达量较高,肝胰腺中表达量最低。在幼体不同蜕壳时期不同部位的肌肉中,Es Akt表达量变化不同,步行足肌肉组织中Es Akt m RNA无显著的统计学差异。腹部肌肉组织中Es Akt m RNA水平在蜕壳前晚期D3~D4期表达量显著高于蜕壳后A^B期和蜕皮间期C期。螯足肌肉在蜕壳前晚期D3~D4期急剧下调,蜕壳后A^B期开始表达量显著升高,直至蜕皮间期C期。研究表明,Es Akt在中华绒螯蟹蜕壳过程中不同部位肌肉组织中的表达量变化与蜕壳周期密切相关,说明Es Akt参与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 c DNA encoding the serine/threonine kinases Akt from Eriocheir sinensis（Es Akt） was cloned by using 3′RACE and 5′RACE techniques, and the sequence and structural analysis of the Es Akt was conducted with bioinformatics methods. The results showed that the full length c DNA encoding Es Akt consisted of 2 200 bp nucleic acids in length, including a 5′-UTR of 159 bp, a 3′-UTR of 496 bp and an open reading frame（ORF） of 1 545 bp encoding 514 amino acids. Analysis of the protein domain features showed that the deduced polypeptides contained three conservative domains characteristic of Serine/Threonine protein kinases family. Multiple sequence alignment revealed that the amnio acids sequences of Es Akt have the 0.889 identity with Fenneropenaeus chinensis and Litopenaeus vannamei. The phylogenetic analysis showed that the Es Akt was arranged in the same clade with Akts from other arthropods. The tissue distribution of Es Akt m RNA in sexual maturity individuals and different muscle groups during molt cycle in juvenile crabs were analyzed by quantitative realtime PCR（q RT-PCR）. In sexual maturity crabs, the Es Akt transcript was detected in eyestalk, claw muscle, ovary,heart, hepatopancreas, epidermis, testis, gill and triangular membrane, and the expression level was relatively high in ovary, eyestalk and testis, and was low in hepatopancreas. In juvenile crabs, the Es Akt transcript in different muscle groups was different depending on the molt stages. In walking leg muscles, the Es Akt expression level has no obvious change. In abdominal muscles, the Es Akt expression level was much higher in later pre-molt D3-D4 stage than post-molt A-B stage and inter-molt C stage. In claw muscles, the Es Akt expression level was decreased rapidly in pre-molt D3-D4 stage and increased in post-molt A-B stage, and lasted to inter-molt C stage. These results suggested that the expression of Es Akt transcript was related with the molt stage of E. sinensis, and it is possible that th

关 键 词：中华绒螯蟹 AKT基因 基因克隆 肌肉生长 蜕壳 

分 类 号：Q785[生物学—分子生物学] S966.16[农业科学—水产养殖]