泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析

程雪艳; 蒋国萍; 柴雪良; 滕爽爽

doi:10.7541/2016.148

泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析

程雪艳^1,2,3,
蒋国萍^1,3,4,
柴雪良^1,2,3,4,
滕爽爽^1,3, ,

1.
浙江省海洋水产养殖研究所, 温州 325005
2.
温州医科大学检验医学院, 生命科学学院, 温州 325035
3.
浙江省近岸水域生物资源开发和保护重点实验室, 温州 325005
3.
浙江省近岸水域生物资源开发和保护重点实验室, 温州 325005
4.
上海海洋大学水产与生命学院, 上海 201306

基金项目:

浙江省重大科技专项 2012C12907-4

国家高技术研究发展计划 2012AA10A410-1

温州市种子种苗科技创新专项 N20120017

详细信息

中图分类号: Q781
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出版历程
- 收稿日期: 2015-12-22
- 修回日期: 2016-05-28
- 发布日期: 2016-10-31

FULL-LENGTH CDNA CLONING AND EXPRESSION ANALYSIS OF GLUTATHIONE PEROXIDASE FROM BLOOD CLAM TEGILLARCA GRANOSA

1.
Zhejiang Mariculture Research Institute, Wenzhou 325005, China;
2.
Laboratory Medicine and Life Science College of Wenzhou Medical University, Wenzhou 325035, China;
3.
Zhejiang Key Laboratory of Exploitation and Preservation of Coastal Bio-resource, Wenzhou 325005, China;
3.
Key Laboratory of Bodogical Resourles Development and Protection of Coastal Wotters of Zhejiang Province, Wenzhou 325005, China;
4.
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

Funds:

Major Science and Technology Projects of Zhejiang Province 2012C12907-4

Supported by National High Technology Research and Development Program of China (863 Program) 2012AA10A410-1

Seed Science and Technology Innovation Project of Wenzhou City N20120017

摘要

摘要: 为了探讨谷胱甘肽过氧化物酶（Glutathione peroxidase，GPx）基因在泥蚶应激反应中的作用，研究采用RACE技术克隆了泥蚶GPx基因（TgGPx）cDNA全长，其cDNA全长1195 bp，包含45 bp 5’-UTR，639 bp开放阅读框（ORF）和511 bp 3’-UTR。ORF编码212个氨基酸残基，预测蛋白分子量为24.3 kD，理论等电点为8.33，其中，第53个氨基酸U是由密码子²⁰²UGA²⁰⁴编码的硒代半胱氨酸（Se-Cys）。在3’-UTR上存在一段序列，形成一种独特的茎环结构，即SECIS元件。SECIS元件在密码子UGA翻译为Se-Cys的过程中起决定性作用。通过序列比对与系统进化分析，发现软体动物中也存在不同种类的GPx基因，TgGPx与GPx1和GPx2的亲缘关系较近。利用qRT-PCR技术对TgGPx在泥蚶的不同组织以及重金属刺激后的表达量进行分析，结果表明，TgGPx在泥蚶的5个组织中都有表达，但存在组织特异性，在外套膜中的表达量最高，在血细胞中的表达量最低。用重金属铅、铜、镉刺激后，TgGPx在肝胰脏中的表达量显著升高，表明TgGPx在维护机体正常功能方面及泥蚶抵御外界刺激的应激反应中发挥作用。
- 泥蚶 /
- 谷胱甘肽过氧化物酶 /
- 全长克隆 /
- 基因表达
Abstract: Glutathione peroxidase (GPx) is an important member of cellular enzymatic antioxidant system regulating stress response of host as an acute protein. In this study, a selenium-dependent glutathione peroxidase (TgGPx) gene from blood clam Tegillarca granosa was cloned and analyzed by rapid amplification of cDNA ends (RACE). The nucleotide sequence of TgGPx was consisted of 1195 bp with a 45 bp 5'UTR a of 511 bp 3'UTR and 639 bp open reading frame (ORF) encoding a peptide of 212 amino acids with an estimated molecular mass of 24.3 kD and a theoretical isoelectric point of 8.33. TgGPx had a characteristic codon at ²⁰²UGA²⁰⁴ that corresponded to Selenocysteine as U53. A selenocysteine insertion sequence (SECIS) element was identified in the 3'-UTR of TgGPx cDNA, which forms a stem-loop secondary structure. SECIS element plays a decisive role in the translation of the stop codon UGA into a selenocysteine. Multiple sequence alignment and phylogenetic analysis revealed that there were different types of GPx genes in mollusks. TgGPx has a closer phylogenic relationship with GPx1 and GPx2. TgGPx expressed in all five tissues with the highest mRNA level in mantle and the lowest in haemocytes. The expression pattern of TgGPx in adult may support its role in adult tissue growth and larval development. TgGPx was significantly up-regulated in hepatopancreas by heavy metals (Zn²⁺, Cu²⁺ and Cd²⁺) exposure, suggesting TgGPx may play a role in stress response and maintaining the body's normal function in T. granosa.
- Tegillarca granosa /
- Glutathione peroxidase /
- Full length clone /
- mRNA expression

HTML全文

图 1 基于不同物种Se-GPx基因氨基酸序列构建的NJ系统进化树

智人GPX1 (H. sapiens GPX1: NP_000572.2); 智人GPX2 (H. sapiens GPX2: NP_002074.2); 智人GPX3 (H. sapiens GPX3: AAP50261.1); 智人GPX4 (H. sapiens GPX4: NP_001034937.1); 小家鼠GPX1 (M. musculus GPX1: AAH86649.1); 小家鼠GPX2 (M. musculus GPX2: NP_109602.2); 小家鼠GPX4 (M. musculus GPX4: NP_001032830.2); 褐家鼠GPX2 (R. norvegicus GPX2: NP_899653.2); 褐家鼠GPX3 (R. norvegicus GPX3: NP_071970.2); 家牛GPx2 (B. taurus GPx2: NP_001156611); 家牛GPx4 (B. taurus GPx4: NP_777195); 斑马鱼GPx1 (D. rerio GPx1: NP_001007282.2); 斑马鱼GPx3 (D. rerio GPx3: NP_001131027.1); 斑马鱼GPx4 (D. rerio GPx4: NP_001007283.2); 三角帆蚌(H. cumingii GPX: ACY72387.1); 南方黑鲔(T. maccoyii GPX: ABO38817.1); 暗纹东方鲀GPX2 (T. obscurus GPX2: ACR20472.1); 曼氏无针乌贼(S. maindroni GPX: AEK48346.1); 虾夷扇贝(M. yessoensis GPX: ADQ92353.1); 真蛸(O. vulgaris GPX: AGZ63440.1); 紫贻贝(M. galloprovincialis GPX: ADY38576.1); 家犬GPX3 (C. lupus familiaris GPX3: NP_001157926.1); 河蚬(C. fluminea GPx: ABQ24217.1); 长牡蛎(C. gigas GPx: ABS19600.1); 斑马贻贝(D. polymorpha GPx: ABP73388.1); 皱纹盘鲍(H. discus hannai GPX: ADD71075.1); 非洲爪蟾GPX4(X. laevis GPX4: NP_001165213.1); 热带爪蟾GPX3 (X. (Silurana) tropicalis GPX3: NP_988961.2); 菲律宾蛤仔(R. philippinarum GPX: ACU83220.1); 文蛤(M. meretrix GPX: ADR51677.1)

Figure 1. Neighbor-joining phylogenetic tree of Se-GPx amino acid sequences from different species

下载: 全尺寸图片幻灯片

图 2 TgGPx在成体泥蚶不同组织的表达

与肝胰脏组织相比, *代表P<0.05; **代表P<0.01; 下同

Figure 2. The expression level of TgGPx in tissues of adult T. granosa

Significant differences across hepatopancreas are indicated with * at P<0.05 and ** at P<0.01; the same applies below

下载: 全尺寸图片幻灯片

图 3 TgGPx基因重金属胁迫后肝胰脏组织的表达情况

Figure 3. The expression level of TgGPx in hepatopancreas after three heavy metal exposures

下载: 全尺寸图片幻灯片

表 1 试验中所用引物序列

Table 1 Primers used in the study

引物Primers	引物序列Primers sequence (5′-3′)
TgGPx-F1	TGCAACCAGTTCGGTCATCAGGAGAA
TgGPx-R1	AGGAACTTCTCAAAGTTCCAGG
TgGPx-F2	ATTAAAGCATGTTCGACC
TgGPx-R2	AGAAATACAAAAAGTGGG
TgGPx-3Race-F1	TTCTACGAGAACGACTGCCGACACCT
TgGPx-3Race-F2	CGACTGCCGACACCTAGTGATGATGC
TgGPx-5Race-F1	CACTAGGTGTCGGCAGTCGTTCTCGTA
TgGPx-5Race-F2	AATCCATTTCCTGGTCGAACATGCTTTA
TgGPx-Real-F	CTTGTGGTTCTGGGGTTCC
TgGPx-Real-R	GTCGGCAGTCGTTCTCGTA
Tg-18S rRNA-F	CTTTCAAATGTCTGCCCTATCAACT
Tg-18S rRNA-R	TCCCGTATTGTTATTTTTCGTCACT

下载: 导出CSV

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FULL-LENGTH CDNA CLONING AND EXPRESSION ANALYSIS OF GLUTATHIONE PEROXIDASE FROM BLOOD CLAM TEGILLARCA GRANOSA

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