香港牡蛎 (Crassostrea hongkongensis) Commd1基因的分子克隆及其在盐度胁迫下的表达分析

doi:10.11978/2016022

摘要/Abstract

摘要：

作为一类新近发现的蛋白家族, COMMD (copper metabolism Murr1 domain) 广泛存在于多种生物体中, 且参与多种生物学过程, 包括钠转运、铜代谢以及核因子kappa B (nuclear factor kappa B, NF-κB)、低氧诱导因子1 (hypoxia inducible factor 1, HIF-1) 的调节等。为研究Commd1在盐度胁迫过程中的作用, 采用RACE技术, 首次从香港牡蛎Crassostrea hongkongensis 中克隆获得Commd1基因cDNA全长序列, 命名为ChCommd1基因。该序列全长为841bp, 5′ 和3′ 非编码区分别为18bp和259bp, 开放阅读框为564bp; 该阅读框编码一个含187个氨基酸, 预测分子量为21.79kDa、理论等电点为5.21的蛋白。同源性比较和系统进化分析表明, 香港牡蛎COMMD1蛋白与太平洋牡蛎Crassostrea gigas、光滑双脐螺Biomphalaria glabrata、加州海兔Aplysia california的同源性较高, 是软体动物COMMD1家族的一个成员。实时荧光定量PCR结果显示, ChCommd1基因在香港牡蛎各胚胎发育时期和成体组织中均有分布, 而且, 在盐度刺激后, 其mRNA表达量在鳃和血淋巴中均明显增加。上述研究结果表明, Commd1基因在香港牡蛎发育和渗透压调节中可能发挥作用, 为深入研究该基因在广盐适应性软体动物中的功能提供了依据。

关键词: 香港牡蛎, Commd1, 基因表达, 盐度胁迫

Abstract:

COMMDs are recently discovered in many multicellular organisms. They are involved in numerous aspects of biological processes, such as the regulation of copper homeostasis, the transport of sodium ions, the activity of the transcription factors NF-κB, and hypoxia-inducible factor (HIF-1). To investigate the role of Commd1 in response to salt stress, we first cloned Commd1 cDNA using Rapid Amplification of the cDNA Ends (RACE) technique in mollusks, Crassostrea hongkongensis (designed ChCommd1). The full-length cDNA is 841 bp containing a 5′-untranslated region (UTR) in 18 bp, a 3′-UTR in 262 bp with a poly (A) tail, and an open reading frame (ORF) in 564 bp; the ORF encodes a 187 amino-acid polypeptide with a predicted molecular mass of 21.79 kDa and an isoelectric point of 5.21. Homologous comparison and phylogenetic analysis revealed that the 187-aa-long ChCOMMD1 protein shares high sequence identity with its homologs from other species and belongs to the molluscan COMMD1 family. Quantitative real-time PCR analysis showed that ChCommd1 mRNA is broadly expressed in various tissues and during different stages of the oyster’s embryonic and larval development. Upon exposure to two stressors (high and low salinity), the expression level of ChCommd1 mRNA increased significantly. Taken together, our results indicated that ChCommd1 can function in the embryonic development and osmotic regulation, providing an important reference for further investigations on the functions of Commd1 in the euryhaline mollusks.

Key words: C. hongkongensis, Commd1, gene expression, salinity stress

王富轩, 肖述, 向志明, 喻子牛. 香港牡蛎 (Crassostrea hongkongensis) Commd1基因的分子克隆及其在盐度胁迫下的表达分析[J]. 热带海洋学报, 2017, 36(1): 48-55.

Fuxuan WANG, Shu XIAO, Zhiming XIANG, Ziniu YU. Molecular cloning and expression analysis of Commd1 under salinity stress in Crassostrea hongkongensis[J]. Journal of Tropical Oceanography, 2017, 36(1): 48-55.

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名称	序列 (5'-3')	用途
ChCommd1-R1	TTGCTGCCTCTCTCCTCTTCATTTG	5'RACE
ChCommd1-R2	ACATCTCTTCAGTAGTGTTGCGTAA
ChCommd1-F1	CGTGTTACAAAATATGAAAGACATT	3'RACE
ChCommd1-F2	GTGCCATAGTTGAACTCCAAATAGA
ChCommd1-F3	GAAAACATGACAAACAAAATGG	ORF扩增
ChCommd1-R3	TGAGTACGGGAATATGATACAA
ChCommd1-F4	AAGCCATTGCTTACAGAAAGTT	荧光定量PCR
ChCommd1-R4	TCAGGGTTTTCTATTTGGAGTT
GAPDH- F	GGATTGGCGTGGTGGTAGAG	内参基因
GAPDH- R	GTATGATGCCCCTTTGTTGAGTC
EF1α-F	CGGGATCCATGTATAGTCGGGAGA	内参基因
EF1α-R	CCCAAGCTTTCACAGAGAAATCAA