Association Analysis And Function Study Of Three Ammonia Tolerance Genes In Sinonovacula Constricta

Ammonia nitrogen accumulation is one of the most common problems in aquatic system,which causes a great threat to the growth,development and even survival of aquaculture animals.In this study,we investigated the effects of short-term acute ammonia stress on three key genes of Glutamate dehydrogenase(Sc-GDH),Glutamine Synthetase(Sc-GS)and Glutathione peroxidase(Sc-GPX)in Sinonovacula constricta,which were involved in glutamine synthesis and response to oxidative stress.First,direct sequencing was used to analyze the association between the polymorphism and ammonia tolerance from two geographical populations.In addition,western blotting(WB),paraffin section and immunofluorescence were used to investigate the expression of Sc-GDH,Sc-GS and Sc-GPX protein expression and theirs histocellular localization in gills and hepatopancreas under short-term acute ammonia stress.Finally,RNA interference(RNAi)technology was used to silence Sc-GDH,Sc-GS and Sc-GPX genes,and the m RNA expression changes of Sc-GDH,Sc-GS and Sc-GPX genes after interference in hepatopancreas were detected by realtime quantitative PCR(q RT-PCR).The results were as follows:1.A total of 26 and 22 single nucleotide polymorphisms(SNPs)of Sc-GDH were identified from Zhejiang(ZJ)and Fujian(FJ)population,respectively.Among them,two SNPs(c.323 T > C,c.620 C > T)exhibited a significant and strong association with ammonia tolerance(P < 0.05).In addition,c.323 T > C,c.620 C > T were synonyms and showed strong linkage disequilibrium.WB results showed that Sc-GDH responded rapidly at 12 h and reached the peak at 24 h after 96 h exposure to 180 mg/L ammonia nitrogen,and then decreased sharply and fluctuated within a certain range.The results of immunofluorescence indicated that the columnar cells of gill filaments and the endothelial cells of hepatopancreas were the major sites for Sc-GDH secretion.Under ammonia stress(180 mg/L),the expression levels of Sc-GDH gene were extremely significantly downregulated at 24,48,72 and 96 h(P < 0.01)after RNAi.In the ammonia stress group,the maximum interference rate of the experimental group was 71.41% compared with the negative control chains(NC),and meanwhile the corresponding value was 74.04% in the non-ammonia stress group.2.The results showed that 12 and 14 SNPs of Sc-GS from ZJ and FJ population were identified,respectively.Among them,one SNP(c.1133 T > G)exhibited a significant and strong association with ammonia tolerance(P < 0.01),which was changed from leucine to arginine(L-R).WB results showed that Sc-GS protein responded rapidly in 0 ～ 6 h and reached a peak at 6 h after 180 mg/L ammonia nitrogen exposure for 96 h,and then fluctuated in a certain range after a short decline at 12 h.The results of immunofluorescence indicated that the columnar cells of gill filaments and the endothelial cells of hepatopancreas were the major sites for Sc-GS secretion.Under ammonia stress(180 mg/L),the expression levels of Sc-GS gene were extremely significantly downregulated at 24,48,72 and 96 h(P < 0.01)after RNAi.In the ammonia stress group,the maximum interference rate of the experimental group was 85.57%compared with the NC,in which non-ammonia stress group the corresponding value efficiency was 84.61%.3.A total of 6 SNPs of Sc-GPX were identified.Among them,two SNPs(c.162 A > C and c.705 T > A)exhibited a significant and strong association with ammonia tolerance(P < 0.01),and c.162 A > C was mutated from lysine to asparagine(K-N).WB results showed Sc-GPX protein responded quickly to ammonia nitrogen stress after 96 h of 140 mg/L ammonia nitrogen exposure.The expression level of ScGPX protein increased rapidly in 3 ～ 6 h and then decreased for a period of time,then increased again at 24 h and remained at a high level for a long time after that.The results of immunofluorescence indicated that the columnar cells of gill filaments and the endothelial cells of hepatopancreas were the major sites for ScGPX secretion.Under ammonia stress(140 mg/L),the expression levels of Sc-GPX gene were extremely significantly downregulated at 24 and 48 h(P < 0.01)after RNAi.In the ammonia stress group,the maximum interference rate of the experimental group was 85.57% compared with the NC,in which non-ammonia stress group the corresponding value efficiency was 84.61%.This will lay a foundation for us to further understand the roles of Sc-GDH,Sc-GS and Sc-GPX genes in ammonia nitrogen detoxification in the future,and provide a reference for the molecular marker-assisted breeding of new varieties in S.constricta resistant to ammonia nitrogen.