| 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。 |
| 关键词: 缢蛏 Na+/K+-ATPase 氨氮排泄 基因表达 RNA干扰 |
| DOI：10.11693/hyhz20230200033 |
| 分类号: |
| 基金项目: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。 |
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| VERIFICATION OF SNP ASSOCIATED WITH AMMONIA TOLERANCE AND EXPRESSION OF SNP-RELATED NKA GENE RESPONDING TO AMMONIA STRESS IN RAZOR CLAM (SINONOVACULA CONSTRICTA) |
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HU Chen-Xin1, LYU Li-Yuan2, SUN Gai-Gai1,3, YAO Han-Han1, LIN Zhi-Hua1,2, DONG Ying-Hui2
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1.Key Laboratory of Aquatic Germplasm Resource of Zhejiang, College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China;2.Ninghai Marine Biological Seed Industry Research Institute, Zhejiang Wanli University, Ningbo 315604, China;3.College of Marine Sciences, Ningbo University, Ningbo 315832, China
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| Abstract: |
| 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. |
| Key words: Sinonovacula constricta Na+/K+-ATPase ammonia excretion gene expression RNA interference |
