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2025年8月13日 星期三
农业生物技术学报  2022, Vol. 30 Issue (11): 2187-2200    DOI: 10.3969/j.issn.1674-7968.2022.11.012
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
红螯光壳螯虾渗透压相关基因的克隆及其在不同盐度下的表达
彭博浩, 张炎, 刘畅, 钟箫, 付德政, 张凯怡, 王艺磊*
集美大学 水产学院,厦门 361021
Cloning of Osmotic Pressure-related Genes from Cherax quadricarinatus and Their Expression Under Different Salinity
PENG Bo-Hao, ZHANG Yan, LIU Chang, ZHONG Xiao, FU De-Zheng, ZHANG Kai-Yi, WANG Yi-Lei*
Fisheries College, Jimei University, Xiamen 361021, China
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摘要 红螯光壳螯虾(Cherax quadricarinatus)是淡水虾,盐度是影响其生长与存活的重要环境因素。为了扩大可用于红螯光壳螯虾养殖的水域,如盐碱地、河口半咸水区,需要了解其渗透压调节机制,为其在半咸水养殖的可行性及通过半咸水暂养改善其风味提供理论依据。本研究通过NCBI已发表的红螯光壳螯虾转录组数据库筛选出3个与渗透压相关基因:钠钾ATP酶(Na+/K+-ATPase, NKA)、钠钾氯共转运体(Na+/K+/2Cl--cotransporter, NKCC)和酪氨酸3-加单氧酶/色氨酸5-加单氧酶激活蛋白(14-3-3),通过RT-PCR验证其ORF区。运用生物信息学在线网站对其蛋白结构进行预测,并将不同物种的同源蛋白序列进行多重序列比对和进化树构建,同时利用qPCR分析NKANKCC14-3-3在红螯光壳螯虾不同组织的表达情况及在不同盐度胁迫24、48和96 h后,在鳃和肝胰腺中的表达变化情况。结果表明,NKA、NKCC、14-3-3蛋白在不同物种间具有较高保守性。NKANKCC14-3-3基因在所检测的红螯光壳螯虾鳃、肌肉、心脏、肝胰腺、眼柄组织中均有表达。在不同盐度胁迫下,鳃组织中的NKA基因的表达量在5‰、10‰盐度下变化不显著,在15‰、20‰盐度组的48 h表达量显著提高(P<0.05),在肝胰腺中NKA基因表达量仅在20‰盐度组的48 h显著上升(P<0.05)。鳃组织中的NKCC基因表达量在10‰、15‰、20‰盐度组的24、48和96 h均显著上调(P<0.05),在肝胰腺中仅在15‰、20‰盐度组的48 h显著上调(P<0.05)。鳃组织中14-3-3基因表达量在10‰、15‰、20‰盐度组的48 h显著上调(P<0.05),在肝胰腺中其表达量在5‰、10‰、15‰盐度组的48 h均显著上调(P<0.05),在20‰盐度组的24和48 h显著上调(P<0.05)。结果表明,NKANKCC14-3-3基因的表达与盐度变化相关,20‰盐度胁迫48 h可能是鳃和肝胰腺在高盐度下渗透调节的关键点。本研究为进一步探讨NKANKCC14-3-3基因在红螯光壳螯虾渗透调节机制中的作用提供了参考依据。
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彭博浩
张炎
刘畅
钟箫
付德政
张凯怡
王艺磊
关键词 红螯光壳螯虾盐度变化钠钾ATP酶(NKA)钠钾氯共转运体(NKCC)酪氨酸3-加单氧酶/色氨酸5-加单氧酶激活蛋白(14-3-3)    
Abstract:Salinity is an important environmental factor affecting the growth and survival of crustaceans. The red claw crayfish (Cherax quadricarinatus) is a freshwater species. To increase the waters available for cultivating this species, saline waters and brackish waters may be potential waters for cultivating this species. Thus, understanding the osmotic regulation mechanism of the red claw crayfish is needed to provide a theoretical basis for farming feasibility in brackish water and improve the flavor by changing the growth environment (temporary culture in brackish water). In this study, 3 genes related to osmolality, Na+/K+-ATPase (NKA), Na+-K+-2Cl--Cotransporter (NKCC) and 14-3-3, were screened by the NCBI published transcriptome database of C. quadricarinatus, and their ORF regions were confirmed by RT-PCR. The protein structure was predicted using an online bioinformatics website, and the homologous protein sequences of different species were subjected to multiple sequence alignment and evolutionary tree construction. The expression of NKA, NKCC and 14-3-3 in different tissues and the changes of expression in gill and hepatopancreas after 24, 48 and 96 h under different salinity in the red claw crayfish were analyzed by qPCR. The results showed that NKA, NKCC and 14-3-3 proteins were highly conserved among different species. NKA, NKCC and 14-3-3 genes were expressed in the gill, muscle, heart, hepatopancreas and eyestalk tissues of red claw crayfish. Under different salinity stresses, the expression of the NKA gene in gill tissues did not change significantly at 5‰ and 10‰ salinity but increased significantly in 15‰ and 20‰ salinity groups at 48 h (P<0.05). In the hepatopancreas, the expression of NKA gene was only significantly increased in the 20‰ salinity group at 48 h (P<0.05). The expression of NKCC gene in gill tissue was significantly up regulated in 10‰, 15‰ and 20‰ salinity groups at 24, 48 and 96 h, and only significantly increased in 15‰ and 20‰ salinity groups at 48 h in hepatopancreas. The expression of the 14-3-3 gene was significantly up regulated in gill tissue in 10‰, 15‰ and 20‰ salinity groups at 48 h (P<0.05), and significantly up regulated in hepatopancreas in 5‰, 10‰ and 15‰ salinity groups at 48 h (P<0.05) and the expression significantly up regulated at 24 and 48 h in 20‰ salinity group (P<0.05). The results showed that the expression of NKA, NKCC and 14-3-3 genes was related to the change of salinity. 20‰ salinity stress after 48 h may be the key point of osmotic regulation of gill and hepatopancreas under high salinity. This study provides a reference for further exploring the role of NKA, NKCC and 14-3-3 genes in the osmoregulatory mechanism of the red claw crayfish.
Key wordsCherax quadricarinatus    Salinity stress    Na+/K+-ATPase (NKA)    Na+-K+-2Cl--Cotransporter (NKCC)    14-3-3
收稿日期: 2021-12-08     
ZTFLH:  S917.4  
基金资助:国家自然科学基金(41676161); 国家级大学生创新创业项目(201810390035S; 202210390003; 202210390030)
通讯作者: * ylwang@jmu.edu.cn   
引用本文:   
彭博浩, 张炎, 刘畅, 钟箫, 付德政, 张凯怡, 王艺磊. 红螯光壳螯虾渗透压相关基因的克隆及其在不同盐度下的表达[J]. 农业生物技术学报, 2022, 30(11): 2187-2200.
PENG Bo-Hao, ZHANG Yan, LIU Chang, ZHONG Xiao, FU De-Zheng, ZHANG Kai-Yi, WANG Yi-Lei. Cloning of Osmotic Pressure-related Genes from Cherax quadricarinatus and Their Expression Under Different Salinity. 农业生物技术学报, 2022, 30(11): 2187-2200.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.11.012     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I11/2187
 
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