草鱼(♀)×赤眼鳟(♂) F1及其亲本CAST基因cDNA全长克隆与结构差异

李东放; 李耀国; 金生振; 何美凤; 肖调义

doi:10.7541/2020.064

草鱼(♀)×赤眼鳟(♂) F₁及其亲本CAST基因cDNA全长克隆与结构差异

李东放^1,,
李耀国^1,2, ,,
金生振¹,
何美凤¹,
肖调义^1,2, ,

1.
湖南农业大学湖南省特色水产资源利用工程技术研究中心, 长沙 410128
2.
水产高效健康生产湖南省协同创新中心, 常德 415000

基金项目: 国家自然科学基金面上项目(31572615); 国家自然基金青年项目(31802288); 中国博士后科学基金特别资助项目(2018T110833); 中国博士后科学基金(2017M612560); 湖南省自然科学基金(2019JJ50231); 湖南农业大学省级优秀博士学位论文培育基金(YB2018002); 湖南省研究生科研创新项目(CX2018B400)资助

详细信息

作者简介:
李东放(1996—), 男, 硕士研究生; 主要研究方向为水产遗传育种与繁殖。E-mail: dfli0927@163.com

通信作者:
李耀国(1986—), 主要研究方向为水产抗病遗传育种。E-mail: yaoguolijkl@163.com

肖调义(1964—), 主要研究方向为水生动物遗传育种。E-mail: tyxiao1128@163.com *共同通信作者

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出版历程
- 收稿日期: 2019-05-15
- 修回日期: 2019-09-04
- 网络出版日期: 2020-05-18
- 发布日期: 2020-04-30

FULL-LENGTH cDNA CLONING AND STRUCTURAL DIFFERENCES OF THE CAST GENE FROM CTENOPHARYNGODON IDELLUS (♀), SQUALIOBARBUS CURRICULUS (♂) AND THEIR HYBRID F₁

1.
Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization, Hunan Agricultural University, Changsha 410128, China
2.
Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Changde 415000, China

Funds: Supported by the National Natural Science Foundation of China (31572615); the NSFC Youth Project (31802288); the Special Support Project of China Postdoctoral Science Foundation (2018T110833); the China Postdoctoral Science Foundation (2017M612560); the Natural Science Foundation of Hunan Province (2019JJ50231); Hunan Agricultural University Provincial Excellent Doctoral Dissertation Cultivation Fund (YB2018002); the Research and Innovation Project for Graduate Students in Hunan Province (CX2018B400)

Corresponding author:

摘要

摘要: 钙蛋白酶抑制蛋白(Calpastatin, CAST)在肌肉生长和肉质特征形成中发挥重要作用。为探究草鱼(Ctenopharyngodon idellus) (♀)×赤眼鳟(Squaliobarbus curriculus) (♂)正交F₁的肉质相关分子基础, 通过RACE(Rapid-amplification of cDNA ends) 技术分别克隆了草鱼(♀)×赤眼鳟(♂) F₁及其亲本的CAST基因cDNA全长, 并利用生物信息学方法分析比较了三种鱼的CAST结构差异。结果表明: 草鱼(♀)、赤眼鳟(♂)及草鱼(♀)×赤眼鳟(♂) F₁的CAST基因cDNA全长分别为3036、3165和3086 bp, 编码901、893和904个氨基酸; 预测蛋白质分子量分别为93.72、92.77和94.02 kD; 推测的理论等电点分别为5.92、6.01和6.02。草鱼(♀)×赤眼鳟(♂) F₁ CAST与草鱼(♀)和赤眼鳟(♂)核苷酸序列相似性分别为94.52%和90%。三种CAST蛋白均包括4个含有典型七肽的钙蛋白酶抑制结构域。草鱼(♀)、赤眼鳟(♂)和F₁ CAST氨基酸残基中分别存在73、82和75个潜在的磷酸化修饰位点。蛋白三级结构分析显示草鱼(♀)、赤眼鳟(♂)和F₁ CAST中分别含有24、12和20个β-折叠, 且均呈链状结构。综合可知, F₁ CAST在序列相似度、磷酸化位点数、蛋白质结构及进化地位与草鱼(♀)均更接近。该研究结果为阐明草鱼(♀)×赤眼鳟(♂)正交F₁肉质形成机理提供了分子基础。
- 草鱼 /
- 赤眼鳟 /
- 杂交F₁ /
- CAST /
- 结构差异
Abstract: Calpastatin plays an important role in muscle growth and meat quality formation. To investigate the molecular basis of the meat quality of hybrid F₁ from Ctenopharyngodon idellus (♀) × Squaliobarbus curriculus (♂), the full-length cDNAs of CiCAST, ScCAST and their hybrid F₁ CAST were cloned by using RACE technology, and structure differences were analyzed by bioinformatics tools in this study. The results showed that the full-length cDNA of CiCAST, ScCAST and hybrid F₁ CAST were 3036, 3165 and 3086 bp in length and encoded 901, 893 and 904 amino acids, respectively; the predicted molecular weights were 93.72, 92.77 and 94.02 kDa, and the theoretical isoelectric points were 5.92, 6.01 and 6.02, respectively. The nucleotide sequence similarity of CAST between F₁ CAST and CiCAST was 94.52% and was 90% between F₁ CAST and ScCAST. The three CAST proteins contained four typical calpain inhibitory domains, which had typical heptapeptide structures. A prediction of the phosphorylation sites showed that there were 73, 82 and 75 potential phosphorylation modification sites in the amino acid residues of CiCAST, ScCAST and F₁CAST, respectively. The CAST tertiary structure analysis showed that CiCAST, ScCAST and F₁CAST contained 24, 12 and 20 β-folds, which were all formed into β-chain structures. It was concluded that F₁ CAST was more similar to CiCAST in sequence similarity, number of phosphorylation sites, protein structure and evolutionary status. The results provide a molecular basis for the elucidation of meat quality formation in hybrid F1 fish.
- Ctenopharyngodon idellus /
- Squaliobarbus curriculus /
- Hybrid F₁ /
- CAST /
- Structural differences

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图 1 CAST蛋白结构域分析

Figure 1. Structural feature analysis of CAST proteins

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图 2 CAST三级结构预测

A、B、C和D分别表示草鱼(♀)、赤眼鳟(♂)、正交F₁和三种结果同一界面组合的三级结构预测图

Figure 2. 3D structure prediction of CAST

The 3D structure prediction results of CiCAST, ScCAST, F₁ CAST and a combination of three 3D structures are represented by A, B, C and D, respectively

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图 3 CAST系统发育树分析

Figure 3. Phylogenetic tree analysis of CAST

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表 1 本文所用的引物序列

Table 1 Primer sequences used in this study

引物名称 Primer name	引物序列 Sequence (5′－3′)	用途 Usage
ScCAST-F	TTCGTTCAGCAGGCATGTCC	扩增中间序列
ScCAST-R	CAATCTATCTGTTTCCGCCCTT	扩增中间序列
F₁CAST-F	GGCACTTTACCATCATCACAACC	扩增中间序列
F₁CAST-R	CAATCTATCTGTTTCCGCCCTT	扩增中间序列
CiCAST5′-RACE	GCCTTAACATCAGGTGCTGCCACG	RACE PCR
CiCAST3′-RACE	CCCACTGTCCCATCTGCTGCCACGACC	RACE PCR
ScCAST5′-RACE	ACCGTGAAGCCCTCCGCACCTGA	RACE PCR
ScCAST3′-RACE	AGATGCCTGCCGAACGAACATCGACT	RACE PCR
F₁CAST5′-RACE	CCGCCCCTGTCCCGCCTAAAGCCAT	RACE PCR
F₁CAST3′-RACE	CTCTCTCCCCGACAAGAACACCC	RACE PCR
注: Ci. 草鱼; Sc. 赤眼鳟; F₁. 草鱼(♀)×赤眼鳟(♂) F₁Note: Ci. Ctenopharyngodon idellus; Sc. Squaliobarbus curriculus; F₁. progeny of Ctenopharyngodon idellus (♀) × Squaliobarbus curriculus (♂)

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