缢蛏（Sinonovacula constricta）GST和HSP90基因克隆及其在氨氮胁迫下的表达特征分析

张欢; 董迎辉; 姚韩韩; 俞凯佳; 胡月亚; 林志华

doi:10.3969/j.issn.0253-4193.2020.04.008

缢蛏（Sinonovacula constricta）GST和HSP90基因克隆及其在氨氮胁迫下的表达特征分析

doi: 10.3969/j.issn.0253-4193.2020.04.008

张欢^{1, 2,},
董迎辉²,
姚韩韩²,
俞凯佳²,
胡月亚²,
林志华^2, ,

1.
宁波大学海洋学院，浙江宁波 315823
2.
浙江万里学院生物与环境学院浙江省水产种质资源高效利用技术研究重点实验室，浙江宁波 315100

基金项目: 国家现代贝类产业技术体系项目（CARS-49）；浙江省农业新品种选育重大科技专项（2016C02055-9）；国家水产种质资源平台运行服务项目（2019DKA30470）；宁波市“环境科学与工程”一流学科项目。

详细信息

作者简介:
张欢（1994-），女，浙江省衢州市人，研究方向为海洋贝类种质资源与遗传育种研究。E-mail：MhuanhuanZ@126.com

通讯作者:
林志华，男，研究员，博士生导师，主要从事贝类遗传育种研究。E-mail：zhihua9988@126.com

中图分类号: P714⁺.5; Q814
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出版历程
- 收稿日期: 2019-05-27
- 修回日期: 2019-08-21
- 网络出版日期: 2020-11-18
- 刊出日期: 2020-04-25

Cloning of GST and HSP90 genes of Sinonovacula constricta and analysis of their expression characteristics under ammonia nitrogen stress

Zhang Huan^{1, 2
,},
Dong Yinghui²,
Yao Hanhan²,
Yu Kaijia²,
Hu Yueya²,
Lin Zhihua^{2
, ,}

1.
School of Marine Sciences, Ningbo University, Ningbo 315823, China
2.
Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China

摘要

摘要: 克隆获得缢蛏(Sinonovacula constricta)谷胱甘肽S-转移酶(Sc-GSTσ)和热休克蛋白90(Sc-HSP90)基因的cDNA全长，分析了它们的组织表达差异及其在氨氮胁迫下的表达特征。结果表明，Sc-GSTσ的全长cDNA为1 414 bp，含有639 bp的开放阅读框(Open Reading Frame，ORF)，编码212个氨基酸，Sc-GSTσ氨基酸序列与其他物种的GST氨基酸序列同源性为31.88%～43.40%；而Sc-HSP90的全长cDNA为2 752 bp，ORF为2 181 bp，编码726个氨基酸，其氨基酸序列与其他物种HSP90的氨基酸序列同源性为76.77%～87.05%。荧光定量PCR分析发现，Sc-GSTσ和Sc-HSP90在缢蛏各组织中均有表达，两者均在肝胰腺中表达量最高。氨氮胁迫后，Sc-GSTσ和Sc-HSP90 mRNA在肝胰腺中表达均显著上调(p<0.05)，表明氨氮胁迫引起机体的应激反应，2个基因可能参与机体解毒或防御过程。但胁迫后期表达量下降推测是机体的防御能力有限，不足以完全保护宿主免受应激诱导的细胞损伤。
- 缢蛏 /
- GST /
- HSP90 /
- 氨氮 /
- 表达特征
Abstract: The full-length cDNA of glutathione S-transferase (Sc-GSTσ) and heat shock protein 90 (Sc-HSP90) genes were cloned from Sinonovacula constricta and their expression characteristics under ammonia nitrogen stress were analyzed. The results show that the full-length cDNA of Sc-GSTσ was 1 414 bp, and containing 639 bp open reading frame (ORF), encoding 212 amino acid polypeptides. The homology of amino acid sequence of Sc-GSTσ with other species’ GST amino acid sequence was 31.88%−43.40%. The full-length cDNA of Sc-HSP90 was 2 752 bp, ORF was 2 181 bp, encoding 726 amino acids. The amino acid sequence was 76.77%−87.05% homology with other species. Quantitative analysis showed that Sc-GSTσ and Sc-HSP90 genes were expressed in all tested tissues, the strongest expression being in the digestive gland. After exposure to ammonia, the mRNA expression of Sc-GSTσ and Sc-HSP90 were significantly up-regulated (p<0.05) in the digestive gland, indicating that ammonia stress induced stress response, both GST and HSP90 may be participate the process of detoxification or defense. However, the decrease of expression in the later period of stress is presumed to be due to the organism have limited ability to defense, which is not enough to protect the host from stress-induced cell damage.
- Sinonovacula constricta /
- GST /
- HSP90 /
- ammonia nitrogen /
- expression characteristics

HTML全文

图 1 Sc-GSTσ结构域和多重比较

a. Sc-GSTσ的结构域与挑选的4种无脊椎动物的GSTσ和3种脊椎动物的前列腺素D合成酶（PGD，A GSTσ）结构域比较；b. 8种动物N末端结构域多重比较；c. 8种动物GSTσ C末端结构域多重比较

Fig. 1 Sc-GSTσ domain structure and multiple sequence alignment

a. The structure domains of Sc-GSTσ were compared with those of four selected other invertebrate GSTs and three vertebrate prostaglandin-D synthases (PGDS, a GSTσ); b. multiple sequence alignment of N-terminal domain from eight animals; c. multiple sequence alignment of GSTσ C-terminal domain from eight animals

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图 2 利用MEGA5.2软件邻接法构建的GST的系统进化树

使用序列的登录号见附录表A1

Fig. 2 The phylogenetic tree of GST constructed by Neighbor-joining method using MEGA5.2 software

Accession numbers of the sequences used in construction of tree are attached in the appendix Table A1

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图 3 Sc-HSP90基因鉴定及系统进化树

a. 8种动物HSP90氨基酸序列多重比较，红色方框为HSP90家族标签，保守序列MEEVD带有下划线；b. 利用MEGA5.2软件采用邻接法构建的HSP90的系统进化树。使用序列的登录号见附录表A2

Fig. 3 Sc-HSP90 gene identified and phylogenetic tree

a. Multiple sequence alignment of HSP90 amino acid sequences from eight animals, HSP90 family signature motifs are red boxes and sequence MEEVD is underlined; b. the phylogenetic tree of HSP90 constructed by Neighbor-joining method using MEGA5.2 software. Accession numbers of the sequences used in construction of tree are attached in the appendix Table A2

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图 4 Sc-GSTσ和Sc-HSP90基因在缢蛏不同组织中及不同氨浓度胁迫后的表达特征

a1. Sc-GSTσ基因的组织表达；a2. 不同氨浓度胁迫后，Sc-GSTσ在缢蛏肝胰腺中的表达量变化；b1. Sc-HSP90基因的组织表达；b2. 不同氨浓度胁迫后，Sc-HSP90在缢蛏肝胰腺中的表达量变化。组织间不同字母的数据差异显著（p<0.05），星号（*）表示在各时间点胁迫组与对照组之间的显著差异（*p<0.05；**p<0.01；***p<0.001）

Fig. 4 The expression characteristics of Sc-GSTσ and Sc-HSP90 genes in different tissues of S. constricta and after different ammonia concentration stress

a1. Expression profile of Sc-GSTσ in S. constricta; a2. expression characteristic analysis of Sc-GSTσ in the digestive gland of S. constricta after ammonia stress; b1. expression profile of Sc-HSP90 in S. contricta; b2. expression characteristic analysis of Sc-HSP90 in the digestive gland of S. contricta data after ammonia stress. Different letters are significantly different among tissues (p<0.05) and the asterisk (*) indicates a significant difference between the stress group and its respective control at different time (*p<0.05; **p<0.01; ***p<0.001)

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表 1 本实验所用引物及序列信息

Tab. 1 The information of primers and sequences used in this experiment

引物名称	序列（5′-3′）	引物信息
Long	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE扩增接头引物
Short	CTAATACGACTCACTATAGGGC	RACE扩增接头引物
T3	GAGAGTGCTCGCAACTTAGAAATCGCCG	Sc-GSTσ 3′RACE扩增
T5	CGGCATGTCTGGTTTCCGTTGG	Sc-GSTσ 5′RACE扩增
P3	CGCATCCCTCAATACTCTTGGTCG	Sc-HSP90 3′RACE扩增
P5	CCTCGTCTTCCTCCAAATCCTCTACCTT	Sc-HSP90 5′RACE扩增
M13 F	CGCCAGGGTTTTCCCAGTCACGAC	T1载体通用引物
M13 R	GAGCGGATAACAATTTCACACAGG	T1载体通用引物
T F1	TGGAGTCTCTGGGTGATACG	Sc-GSTσ全长验证引物
T R1	ACAACAACGCTGAAACTGGA	Sc-GSTσ全长验证引物
T F2	TTCTTGTGGAGCAGTACAGTTCTAA	Sc-GSTσ全长验证引物
T R2	CAAAATCAAAAGGCTGTCGC	Sc-GSTσ全长验证引物
P F1	GTTTACTCAGAAAGACGCACGG	Sc-HSP90全长验证引物
P R1	CTACTACGACGCATAATGGAGC	Sc-HSP90全长验证引物
P F2	GGCTGCCAAGAAGAACCTA	Sc-HSP90全长验证引物
P R2	AAATCACAGATTGAACAATCAAGTA	Sc-HSP90全长验证引物
RS9 F	TGAAGTCTGGCGTGTCAAGT	内参基因荧光定量引物
RS9 R	CGTCTCAAAAGGGCATTACC	内参基因荧光定量引物
GST F	GAACCGACGAGAATCAGAAGA	Sc-GSTσ荧光定量引物
GST R	AAACATCGCCGAAACCACG	Sc-GSTσ荧光定量引物
HSP90 F	GATGAAGGGGAGGTGGAAA	Sc-HSP90荧光定量引物
HSP90 R	GTGTCAATGAGGGTCAGTGTGT	Sc-HSP90荧光定量引物

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