瓦氏雅罗鱼<em>Commd9</em>基因的克隆及其在碱度胁迫下的表达分析

doi:10.3969/j.issn.1674-7968.2022.09.011

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摘要铜转运Murr1结构域(copper metabolism Murr1 domain, COMMD)蛋白家族在生物体中广泛存在,参与机体核因子κB (nuclear factor-κB, NF-κB)活性调节、钠转运、铜代谢以及缺氧的适应性调节等生物学过程。为研究Commd9在碱度胁迫下的作用,本研究采用cDNA末端快速扩增(rapid-amplification of cDNA ends, RACE)技术,首次对具有耐高碱特性的瓦氏雅罗鱼(Leuciscus waleckii) Commd9基因cDNA全长进行了克隆,命名为LwCommd9基因(GenBank No. MT013199)。该序列全长为938 bp,5'和3'非编码区分别为5和339 bp,开放阅读框为594 bp,编码蛋白含197个氨基酸,预测分子量为21.79 kD,理论等电点为5.63。系统发育分析显示,瓦氏雅罗鱼COMMD9氨基酸序列与鲤科鱼类的相似性较高,其中与鱇浪白鱼(Anabarilius grahami)相似性高达93.37%。qPCR发现,LwCommd9基因在各组织中均有分布,在肝和肾组织中表达量显著高于其他组织(P<0.05)。高碳酸盐碱度胁迫下,LwCommd9基因在碱水种群瓦氏雅罗鱼肝组织中的表达量极显著下调(P<0.01),而在淡水种群瓦氏雅罗鱼肝组织中的表达量极显著上调(P<0.01),在肾组织中也有相同变化趋势。综上所述,LwCommd9基因在瓦氏雅罗鱼高碱适应过程中可能起到了关键作用,这将为后续鱼类耐高碱机制研究提供参考。

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关键词 ：瓦氏雅罗鱼, 铜转运Murr1结构域9 (Commd9)基因, 基因表达, 碱度胁迫

Abstract：The copper metabolism Murr1 domain (COMMD) protein family is an important biologic part of the nuclear factor-κB (NF-κB) pathway, sodium transport, copper regulation, and adaptive regulation in hypoxia. Commd9 cDNA in high-alkaline-tolerant fish, Leuciscus waleckii, was cloned firstly and given the name LwCommd9 (GenBank No. MT013199) by rapid-amplification of cDNA ends (RACE) to find out what Commd9 does in alkaline environment. The results showed that the full-length cDNA sequence of the Commd9 gene was 938 bp in L. waleckii, which had 5 bp of 5' non-coding region, 339 bp of 3' non-coding region, and 594 bp of the ORF fragment could code for 197 amino acids that had a predicted molecular weight of about 21.79 kD and a theoretical isoelectric point of 5.63. Phylogenetic analysis revealed that the amino acid sequence of LwCommd9 showed high similarity with carp, particularly with Anabarilius grahami up to 93.37%. qPCR showed that LwCommd9 were expressed in virous organs, such as significant increase in liver and kidney (P<0.05). In addition, LwCommd9 expression in liver was significantly down regulated (P<0.01) in alkaline water species (alkali-water, AW), but that was highly up regulated (P<0.01) in freshwater species (FW) and showed in kidney with the same trend. The above results indicate that the Commd9 gene in L. waleckii might play a critial role in the process of high-alkaline adaptation, and the in-depth mechanisms related to this pathway can be explored deeply.

Key words： Leuciscus waleckii Copper metabolism Murr1 domain 9 gene (Commd9) Gene expression Alkalinity stress

收稿日期: 2022-03-15

ZTFLH:

S917.4

基金资助:国家重点研发计划项目(2020YFD0900402); 黑龙江省自然科学基金青年项目(QC2018038); 中国水产科学研究院基本科研业务费项目(2020TD56); 农业农村部淡水水产生物技术与遗传育种重点实验室开放课题(FBB2017-04)

通讯作者: *jchen1975@163.com;liangliqun@hrfri.ac.cn

引用本文:

罗亮, 常玉梅, 孙博, 张立民, 陈炯, 梁利群. 瓦氏雅罗鱼Commd9基因的克隆及其在碱度胁迫下的表达分析[J]. 农业生物技术学报, 2022, 30(9): 1771-1782.
LUO Liang, CHANG Yu-Mei, SUN Bo, ZHANG Li-Min, CHEN Jiong, LIANG Li-Qun. Cloning of Commd9 Gene in Amur ide (Leuciscus waleckii) and Its Expression Analysis Under Alkalinity Stress. 农业生物技术学报, 2022, 30(9): 1771-1782.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.09.011 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I9/1771

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