缢蛏(Sinonovacula constricta)耐氨氮关联SNP验证及相关基因NKA在氨氮胁迫下的表达特征分析  

VERIFICATION OF SNP ASSOCIATED WITH AMMONIA TOLERANCE AND EXPRESSION OF SNP-RELATED NKA GENE RESPONDING TO AMMONIA STRESS IN RAZOR CLAM(SINONOVACULA CONSTRICTA)

作　　者：胡晨馨 吕丽媛 孙改改 姚韩韩[1] 林志华[1,2] 董迎辉[2] HU Chen-Xin;LYU Li-Yuan;SUN Gai-Gai;YAO Han-Han;LIN Zhi-Hua;DONG Ying-Hui(Key Laboratory of Aquatic Germplasm Resource of Zhejiang,College of Biological and Environmental Sciences,Zhejiang Wanli University,Ningbo 315100,China;Ninghai Marine Biological Seed Industry Research Institute,Zhejiang Wanli University,Ningbo 315604,China;College of Marine Sciences,Ningbo University,Ningbo 315832,China)

机构地区：[1]浙江万里学院生物与环境学院、浙江省水产种质资源高效利用技术研究重点实验室,浙江宁波315100 [2]浙江万里学院宁海海洋生物种业研究院,浙江宁波315604 [3]宁波大学海洋学院,浙江宁波315832

出　　处：《海洋与湖沼》2023年第5期1413-1423,共11页Oceanologia Et Limnologia Sinica

基　　金：国家自然科学基金项目,32202918号;浙江省水产新品种选育重大科技专项课题,2021C02069-7号;宁波市“科技创新2025”重大专项,2021Z114号,2019B10005号。

摘　　要：为筛查和验证缢蛏(Sinonovacula constricta)耐氨氮相关单核苷酸多态性(SNP)和基因,为分子标记辅助育种提供参考,通过竞争性等位基因特异性PCR(KASP)对位于缢蛏NKA基因(Sc-NKA)下游的SNP(g.21062868T>A)进行验证,采用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(WB)检测高氨氮胁迫下Sc-NKA基因的表达水平,用免疫荧光技术对Sc-NKA蛋白进行组织细胞定位,通过RNA干扰探究Sc-NKA基因在氨氮排泄中的作用。结果显示,SNP位点(g.21062868T>A)与氨氮耐受性状显著相关(P<0.05);Sc-NKA基因在氨氮胁迫后的mRNA和蛋白表达量均显著增加(P<0.05);缢蛏鳃中的柱状细胞和扁平细胞是Sc-NKA蛋白分泌的主要部位,且在氨氮胁迫后细胞中的蛋白丰度增加;干扰Sc-NKA基因6~48 h后,缢蛏血淋巴中氨含量显著升高(P<0.05),且Na^(+)-K^(+)-2Cl共转运蛋白1(NKCC1)的mRNA表达水平显著降低(P<0.05)。这些研究结果表明,Sc-NKA参与氨氮的排泄过程,且与NKCC1在氨氮转运中具有协同作用。To screen and verify the single nucleotide polymorphisms(SNPs)and genes that are related to ammonia tolerance of Sinonovacula constricta to guide the molecular marker-assisted breeding,the SNP(g.21062868T>A)located at the downstream of the NKA gene in S.constricta(Sc-NKA)was validated by competitive allele-specific PCR(KASP)analysis.The expression of Sc-NKA gene under high ammonia stress was detected by quantitative real-time PCR(qRT-PCR)and the western blotting.The tissue localization of Sc-NKA protein was carried out using immunofluorescence,and the function of Sc-NKA gene in ammonia excretion was analyzed via RNA interference.The results show that the SNP(g.21062868T>A)was significantly associated with ammonia tolerance(P<0.05).The expression of Sc-NKA significantly increased under ammonia stress(P<0.05).In addition,the columnar cells and flat cells in the gill of S.constricta were the main sites of Sc-NKA protein secretion,and the protein abundance in these cells increased under ammonia stress.Furthermore,the hemolymph ammonia concentration was significantly increased in 6~48 h after the interference of Sc-NKA(P<0.05),with a significant decrease of the mRNA expression of Na^(+)-K^(+)-2Cl cotransporter 1(NKCC1)(P<0.05).This study demonstrated that Sc-NKA was involved in ammonia excretion,and NKA and NKCC1 might have synergism in ammonia transport.

关 键 词：缢蛏 Na^(+)/K^(+)-ATPase 氨氮排泄 基因表达 RNA干扰 

分 类 号：S968.3[农业科学—水产养殖] Q953[农业科学—水产科学] Q789[生物学—动物学]