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
作者简介:王富轩(1988—), 男, 山东省菏泽市人, 硕士研究生, 主要从事贝类免疫与抗逆的研究。E-mail:wangfuxuanzi@163.com
收稿日期: 2016-03-01
要求修回日期: 2016-08-26
网络出版日期: 2017-01-19
基金资助
国家自然科学基金项目(31572661、31202021、31272658)
广州市科技计划项目(201605120657132)
Molecular cloning and expression analysis of Commd1 under salinity stress in Crassostrea hongkongensis
Received date: 2016-03-01
Request revised date: 2016-08-26
Online published: 2017-01-19
Supported by
National Natural Science Foundation of China (31572661, 31202021, 31272658)
Science and Technology Program of Guangzhou, China (201605120657132)
Copyright
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。
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 . DOI: 10.11978/2016022
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
Tab. 1 Sequences of designed primers used in this study表1 引物信息 |
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 |
注: F表示正向引物, R表示反向引物 |
Fig. 1 Nucleotide and amino acid sequences of ChCommd1图1 ChCommd1 全长cDNA核苷酸序列及其编码的氨基酸序列 |
Fig. 2 Multiple alignment of the putative amino acid sequence of ChCommd1 with its homologues in other organisms图2 香港牡蛎与其他物种COMMD1氨基酸序列的多重比对 |
Fig. 3 Maximum-likelihood phylogenetic tree of ChCommd1 amino acid sequences图3 基于ML法构建的ChCOMMD1系统进化树 |
Fig. 4 Expression profiles of ChCommd1 during different stages of oyster’s embryonic and larval development图4 ChCommd1在不同胚胎发育时期的表达模式 |
Fig. 5 Expression profiles of ChCommd1 in different adult tissues图5 ChCommd1在成贝不同组织中的表达模式 |
Fig. 6 Expression profiles of ChCommd1 in gill tissues under high salinity stress and low salinity stress图6 高盐和低盐胁迫下ChCommd1在腮组织中的表达模式 |
Fig. 7 Expression profiles of ChCommd1 in hemocytes under high salinity stress and low salinity stress图7 高盐和低盐胁迫下ChCommd1在血淋巴中的表达模式 |
The authors have declared that no competing interests exist.
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