马氏珠母贝水通道蛋白基因AQP4 cDNA克隆和表达分析

doi:10.11978/2019074

热带海洋学报 ›› 2020, Vol. 39 ›› Issue (3): 66-75.doi: 10.11978/2019074CSTR: 32234.14.2019074

马氏珠母贝水通道蛋白基因AQP4 cDNA克隆和表达分析

潘肖兰^1,^2,³, 刘惠茹^1,^2,³, 许濛^1,^2,³, 许瀚之^1,^2,³, 张华^1,⁴, 何毛贤^1,⁴()

^1. 中国科学院热带海洋生物资源与生态重点实验室, 中国科学院南海海洋研究所, 广东广州 510301
^2. 广东省应用海洋生物学重点实验室, 广东广州 510301
^3. 中国科学院大学, 北京 100049
^4. 中国科学院南海生态环境工程创新研究院, 广东广州 510301

收稿日期:2019-08-19 修回日期:2019-11-28 出版日期:2020-05-10 发布日期:2020-05-19
通讯作者: 何毛贤
作者简介:潘肖兰(1996—), 女, 广西壮族自治区河池市人, 硕士研究生, 研究方向为马氏珠母贝分子遗传育种。E-mail:panxiaolan17@mails.ucas.edu.cn
基金资助:
国家贝类产业技术体系岗位科学家(CARS-49)(CARS-49);海洋生态文明工程项目(ISEE2018PY03);广东省科技计划项目(2017B0303014052)

Cloning and expression analysis of aquaporin gene AQP4 cDNA from Pinctada fucata martensii

Xiaolan PAN^1,^2,³, Huiru LIU^1,^2,³, Meng XU^1,^2,³, Hanzhi XU^1,^2,³, Hua ZHANG^1,⁴, Maoxian HE^1,⁴()

^1. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
^2. Guangdong Provincial Key Laboratory of Applied Marine Biology, Guangzhou 510301, China
^3. University of Chinese Academy of Sciences, Beijing 100049, China
^4. Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou 510301, China

Received:2019-08-19 Revised:2019-11-28 Online:2020-05-10 Published:2020-05-19
Contact: Maoxian HE
Supported by:
The Earmarked Fund for Modern Agro-industry Technology Research System(CARS-49);Marine Ecological Civilization Project(ISEE2018PY03);Science and Technology Planning Project of Guangdong Province, China(2017B0303014052)

摘要/Abstract

摘要：

水通道蛋白4(aquaporin4, AQP4)是一类被动运输水的通道蛋白。本研究根据转录组测序获得的马氏珠母贝AQP4片段, 采用RACE技术获得了AQP4的全长cDNA, 命名为PfAQP4, 其5′非翻译区(untranslated region, UTR)为114bp, 3′UTR为839bp, 开放阅读框(open reading frame, ORF)为858bp, 编码285个氨基酸。PfAQP4含有6个α螺旋跨膜区、5个环、1个主要内嵌蛋白(major intrinsic protein, MIP)结构域、2个NPA(Asn-Pro-Ala)基序, 属于AQP1-like型。利用qPCR技术分析PfAQP4在不同组织、不同发育时期以及盐度胁迫条件下的mRNA表达模式。结果显示: (1) PfAQP4在所有被检测组织中均表达, 其中在闭壳肌、足和鳃中表达量较高; (2) PfAQP4在不同的发育时期均有表达, 其表达量整体呈现先升高后降低的趋势, 其中在2—4细胞期表达量最高, 在眼点幼虫期表达量最低; (3) 在高盐度组(36‰)盐度胁迫24、72、120h后, PfAQP4表达量显著上升, 在168h降至对照组水平; 在低盐(16‰)组, PfAQP4表达量在24h显著上调, 在72h恢复至对照组水平, 说明盐度影响鳃组织中PfAQP4的表达量, 也表明PfAQP4在马氏珠母贝渗透压调节中起着非常重要的作用。

关键词: 马氏珠母贝, 水通道蛋白4, 盐度胁迫, 渗透压调节

Abstract:

Aquaporin 4 (AQP4) is a major channel that passively transports water. In this study, based on a fragment of AQP4 of Pinctada fucata martensii obtained by transcriptome sequencing, the full cDNA was obtained using RACE technology, named PfAQP4, which includes 114 bp of 5'UTR, 839 bp of 3'UTR, and 858 bp of the Open Reading Frame (ORF) encoding a total of 285 amino acids. The PfAQP4 has six alpha-helix transmembrane regions, five loops, one major intrinsic protein (MIP) domain, and two NPAs (Asn-Pro-Ala) motif, indicating that it belongs to AQP1-like type. Real-time fluorescent quantitative PCR was used to analyze the mRNA expression pattern of PfAQP4 in different tissues, different developmental stages, and different salinity stress conditions. The results showed that: (1) PfAQP4 was expressed in all tested tissues, and its expression level was higher in adductor muscle, foot, and gill; (2) in different developmental stages, PfAQP4 was increased first and then decreased, highly expressed in the 2-4 cell stage and lowly expressed in eye-spot larva stage; (3) the expression of gill PfAQP4 mRNA in hypersaline group (36‰) increased significantly at 24h, 72h, and 120h, and returned to the control level at 168 h; in the hyposaline group (16‰), PfAQP4 was significantly up-regulated at 24 h, and the expression level returned to the control level at 72h. These results showed that the salinity can affect the expression of PfAQP4 in gill, and PfAQP4 had a very important effect on the osmoregulation of P. f. martensii.

Key words: Pinctada fucata martensii, aquaporin 4, salinity stress, osmoregulation

潘肖兰, 刘惠茹, 许濛, 许瀚之, 张华, 何毛贤. 马氏珠母贝水通道蛋白基因AQP4 cDNA克隆和表达分析[J]. 热带海洋学报, 2020, 39(3): 66-75.

Xiaolan PAN, Huiru LIU, Meng XU, Hanzhi XU, Hua ZHANG, Maoxian HE. Cloning and expression analysis of aquaporin gene AQP4 cDNA from Pinctada fucata martensii[J]. Journal of Tropical Oceanography, 2020, 39(3): 66-75.

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引物名称	引物序列(5′—3′)	注释
GSP5	CGATAGCCCAAAGGCAAAAGATA	5′RACE外引物
NGSP5	GAAGGTGGCAGAGGTACTCTTGAAGG	5′RACE内引物
GSP3	GTTCGTCTTTGCTGCTCCTTCC	3′RACE外引物
NGSP3	GACCTGAAGAAGGACGAAAACG	3′RACE内引物
Faqp	CTGTGATGGCGTCAACTGATG	PfAQP4定量引物
Raqp	CGCAATCGTGAGAAGATGACC	PfAQP4定量引物
F18S	CGTTTCAACAAGACGCCAGTAG	内参基因18S定量引物
R18S	ACGAAAAAAAGGTTTGAGAGACG	内参基因18S定量引物

物种名	多序列比对用缩写名	登录号	一致性/%
马氏珠母贝(Pinctada fucata martensii)	P.f	MN310685	100
斑马鱼(Danio rerio)	D.r	ACV73794.1	39.5
鱇浪白鱼(Anabarilius grahami)	A.g	ROL42342.1	37.9
斑点叉尾鮰(Ictalurus punctatus)	I.p	AHH42521.1	40.9
暗纹东方鲀(Takifugu obscurus)	T.o	BAL44699.1	40.3
小鼠(Mus musculus)	M.m	AAC53155.1	40.4
大鼠(Rattus norvegicus)	R.n	2D57	41.1
智人(Homo sapiens)	H.s	3GD8	42.2
非洲爪蟾(Xenopus tropicalis)	X.t	NP_001304774.1	39.9
长牡蛎(Crassostrea gigas)	C.g	EKC31493.1	53.4
虾夷扇贝(Mizuhopecten yessoensis)	M.y	OWF48037.1	52.2
停乳链球菌(Streptococcus dysgalactiae)		SQB67194.1	23.4
犬链球菌(Streptococcus canis)		VEE24698.1	23.5

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马氏珠母贝水通道蛋白基因AQP4 cDNA克隆和表达分析

Cloning and expression analysis of aquaporin gene AQP4 cDNA from Pinctada fucata martensii

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