缢蛏多巴胺受体基因及其在损伤修复中的作用

doi:10.11978/2017068

热带海洋学报 ›› 2018, Vol. 37 ›› Issue (3): 45-54.doi: 10.11978/2017068CSTR: 32234.14.2017068

缢蛏多巴胺受体基因及其在损伤修复中的作用

杜蕴超¹(), 谢淑媚¹, 何圣耀¹, 牛东红^1,²(), 李家乐^1,³

1. 上海海洋大学, 水产种质资源发掘与利用教育部重点实验室, 上海 201306
2. 上海海洋大学, 水产科学国家级实验教学示范中心, 上海 201306
3. 上海海洋大学, 上海水产养殖工程技术研究中心, 上海 201306

收稿日期:2017-06-09 修回日期:2018-01-02 出版日期:2018-06-10 发布日期:2018-05-03
作者简介:
作者简介：杜蕴超(1993—), 女, 山东省威海市人, 硕士, 研究方向: 贝类种质资源与遗传育种。E-mail:dorayunc@qq.com
基金资助:
国家自然科学基金项目(31472278);国家高技术研究发展计划(2012AA10A400-3)

Dopamine receptor genes of Sinonovacula constricta and its functions in damage healing of tissue

Yunchao DU¹(), Shumei XIE¹, Shengyao HE¹, Donghong NIU^1,²(), Jiale LI^1,³

1. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China
2. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
3. Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai 201306, China

Received:2017-06-09 Revised:2018-01-02 Online:2018-06-10 Published:2018-05-03
Supported by:
National Basic Research Program of China (31472278);National High-tech R & D Program (“863” Program) of China (2012AA10A400-3)

摘要/Abstract

摘要：

多巴胺受体(dopamine receptor)作为生物体中重要的神经递质受体, 在生物体的生长、发育、代谢等多个生理过程中都发挥着重要的功能。本研究从缢蛏Sinonovacula constricta转录组文库中筛选获得多巴胺受体基因的部分片段, 结合RACE(rapid-amplification of cDNA ends)技术和降落 PCR(polymerase chain reaction)技术, 克隆得到缢蛏两个多巴胺受体的选择性多聚腺苷酸化(alternative polyadenylation, APA)的变异体, 命名为ScDopR2a和 ScDopR2b, 长度分别为1824bp和2758bp。两个变异体均包含相同的5′非翻译区(untranslated regions, UTR)(24bp)和开放阅读框(open reading frame, ORF)(1440bp), 共编码479个氨基酸残基。但是两个变异体的3′UTR 长度不同, 分别为360bp和1294bp, 其中ScDopR2b在polyA尾前插入936bp。在同源的ORF区设计引物, 采用实时定量PCR分析多巴胺受体基因在不同组织中的表达特征, 结果表明多巴胺受体基因在水管、鳃、斧足的表达量显著高于其他组织。在该基因序列3′UTR插入片段区域设计特异引物检测ScDopR2b的组织表达情况, 结果表明在水管、鳃、斧足的表达量仍高于其他组织, 表达趋势与ORF区大致相同。进一步设计缢蛏组织损伤实验, 对进水管前端进行损伤处理, 在处理后的第4h、8h、12h、24h、48h、60h和72h取样, 荧光定量检测结果显示, 同源区表达量在12h和48h呈上调, 在8h、24h和72h下调, 且ScDopR2b表达趋势与同源区表达模式大致相同。研究结果表明, 缢蛏多巴胺受体参与了损伤修复过程, 其表达特征可能与多巴胺作为补偿性神经递质的作用有关。

关键词: 缢蛏, 多巴胺受体, 选择性多聚腺苷酸化, 损伤修复

Abstract:

Dopamine receptor is an important neurotransmitter receptor, playing a key role in organism growth, development, metabolism, and other physiological processes. In this study, we obtained a fragment of dopamine receptor gene from transcriptome library of razor clam (Sinonovacula constricta), and cloned the full-length cDNA of dopamine receptor gene using RACE (rapid-amplification of cDNA ends) and Touchdown PCR (polymerase chain reaction). The full-length cDNA sequence contains two isoforms of alternative polyadenylation (APA), named as ScDopR2a and ScDopR2b, with a length of 1824 bp and 2758 bp, respectively. Both isoforms contain a 24 bp 5′untranslated region (UTR), and a 1440 bp open reading frame encoding 479 amino acid residues. However, the 3′UTR length is 360 bp and 1294 bp for ScDopR2a and ScDopR2b, respectively, with an insertion of 936 bp in front of the polyA tail for ScDopR2b. Primers were designed in open reading frame (ORF)to detect the expression level of dopamine receptor gene (ScDopR2) by quantitative real-time PCR. The results revealed the expression in waterpipe, gill and foot were higher than that in the other tissues. Then, primers were designed in 3′UTR to detect the expression level of ScDopR2b, showing similar expression pattern with ORF region. In the tissue damage experiment, the samples were selected at 4, 8, 12, 24, 48, 60, and 72 h after injuring waterpipe. The fluorescence quantitative results showed that a significant increase in 12 and 48 h, and a significant decrease in 8, 24 and 72 h. The trend of ScDopR2b gene’s expression level was similar to that of ORF. The results suggested that dopamine receptors are involved in the damage-healing process, and the expression pattern may be due to a role of dopamine as compensatory neurotransmitters.

Key words: Sinonovacula constricta, dopamine receptor, alternative polyadenylation (APA), damage healing

杜蕴超, 谢淑媚, 何圣耀, 牛东红, 李家乐. 缢蛏多巴胺受体基因及其在损伤修复中的作用[J]. 热带海洋学报, 2018, 37(3): 45-54.

Yunchao DU, Shumei XIE, Shengyao HE, Donghong NIU, Jiale LI. Dopamine receptor genes of Sinonovacula constricta and its functions in damage healing of tissue[J]. Journal of Tropical Oceanography, 2018, 37(3): 45-54.

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引物	序列(5′-3′)	用途
F	ATCCACAGTGCTACAAGAGTTGC	中间片段扩增
R	CTGCTGACGACGTCTGTTCT	中间片段扩增
3R1	AGTGAAAGTGACGAGGACGACCATACCAAC	3′RACE
3R2	TTCCAACCACGAGGTGCAAACAGAAGCTAC	3′RACE
3R3	ATCAACATCACAACAATCAGCGACCAATG	3′RACE
3R4	TTCATCACCAAACGTCTCCGACAATTAGC	3′RACE
3R5	TACAGATTCTACCAGAACAGACGTCGTCAGC	3′RACE
下游内部引物	CGCGGATCCTCCACTAGTGATTTCACTATAGG	3′RACE
下游外部引物	TACCGTCGTTCCACTAGTGATTT	3′RACE
18SF	TCGGTTCTATTGCGTTGGTTTT	荧光定量内参引物
18SR	CAGTTGGCATCGTTTATGGTCA	荧光定量内参引物
ScdoprF	CTTCCAATCGGCTGTTCG	ORF区段荧光定量引物
ScdoprR	GGTATGGTCGTCCTCGTCAC	ORF区段荧光定量引物
ScDopR2bF	AACTGTCCGTAACGCCAAAG	ScDopR2b特异区荧光定量引物
ScDopR2bR	TCAAACATCCCACTTCGGG	ScDopR2b特异区荧光定量引物
ORF-F	CATCACAACAATCAGCGAC	半定量验证引物
ORF-R	ACAACAATCCCAAGCGTC	半定量验证引物
UTR-F	TTCAGCGTGGTTGATGTG	半定量验证引物
UTR-R	CTTTGGCGTTACGGACAGT	半定量验证引物

位置/bp	PAS序列	分值
1720	TATAAA	0.469
1727	TATAAA	0.577
1794	TATAAA	0.558
2735	AATAAA	0.097

基因名	物种名	登录号
AaDopR1	Aedes aegypti	AFB73766.1
BmDopR1	Bombyx mori	NP_001108459.1
MyDopR1	Mizuhopecten yessoensis	XP_021361185.1
MsDopR1	Manduca sexta	AEU17117.1
LfDopR2	Lampetra fluviatilis	ADO23655.3
HsDopR2	Homo sapiens	NP_000786.1
MyDopR2	Mizuhopecten yessoensis	XP_021340507.1
BgDopR2	Biomphalaria glabrata	XP_013063747.1
CvDopR2	Crassostrea virginica	XP_022345762.1
CgDopR2	Crassostrea gigas	XP_019919825.1
AcDopR2	Aplysia californica	XP_005099999.1
RmDopR2	Rhipicephalus microplus	AFC88980.1
CsDopR2	Chilo suppressalis	AKR18179.1
MmDopR2	Mus musculus	NP_034207.2
BmDopR2	Bombyx mori	NP_001108338.1
ObDopR2	Octopus bimaculoides	XP_014768037.1
LaDopR2	Lingula anatina	XP_013419306.1
LpDopR2	Limulus polyphemus	XP_013783833.1

缢蛏多巴胺受体基因及其在损伤修复中的作用

Dopamine receptor genes of Sinonovacula constricta and its functions in damage healing of tissue

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