中华绒螯蟹Akt基因的cDNA克隆、序列分析及表达特征
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1. 广东韶关学院英东生命科学学院,广东韶关学院英东生命科学学院,上海海洋大学 农业部淡水水产种质资源重点实验室,上海海洋大学 农业部淡水水产种质资源重点实验室

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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)


Molecular cloning, sequence analysis and tissue expression of serine/threonine kinases Akt from Eriocheir sinensis
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College of Yingdong Life Science,Shaoguan University,,Key Laboratory of Freshwater Fishery Germplasm Resources of Ministry of Agriculture, Shanghai Ocean University,

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    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EsAkt)的全长为2 200 bp的cDNA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EsAkt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,EsAkt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RT-PCR技术分析EsAkt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,EsAkt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,EsAkt表达量变化不同,步行足肌肉组织中EsAkt mRNA无显著的统计学差异。腹部肌肉组织中EsAkt mRNA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,EsAkt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EsAkt参与中华绒螯蟹蜕壳诱导的肌肉萎缩、生长及重建过程。

    Abstract:

    In the present study, full length cDNA encoding the serine/threonine kinases Akt from Eriocheir sinensis (EsAkt) was cloned by using 3'RACE and 5'RACE techniques, and the sequence and structural analysis of the EsAkt was conducted with bioinformatics methods. The results showed that the full length cDNA encoding EsAkt 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 EsAkt have the 0.889 identity with Fenneropenaeus chinensis and Litopenaeus vannamei. The phylogenetic analysis showed that the EsAkt was arranged in the same clade with Akts from other arthropods. The tissue distribution of EsAkt mRNA in sexual maturity individuals and different muscle groups during molt cycle in juvenile crabs were analyzed by quantitative real-time PCR (qRT-PCR). In sexual maturity crabs, the EsAkt 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 EsAkt transcript in different muscle groups was different depending on the molt stages. In walking leg muscles, the EsAkt expression level has no obvious change. In abdominal muscles, the EsAkt 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 EsAkt 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 EsAkt transcript was related with the molt stage of E. sinensis, and it is possible that the EsAkt is involved in the muscle atrophy, growth and rebuilding during the molt cycle of E. sinensis.

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田志环,焦传珍,成永旭,吴旭干.中华绒螯蟹Akt基因的cDNA克隆、序列分析及表达特征[J].水产学报,2018,42(4):485~494

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  • 收稿日期:2017-01-08
  • 最后修改日期:2017-04-04
  • 录用日期:2017-07-17
  • 在线发布日期: 2018-03-28
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