Preliminary Investigation On The Mechanism Of Macrobrachium Nipponense In Response To High Salinity Stress Based On Omics

Macrobrachium nipponense,commonly known as river shrimp and prawn,is one of the most high-economic aquaculture varieties in China,which is widely distributed in the freshwater of the country.Due to the limited natural fishing quantity of M.nipponense,large-scale and intensive artificial breeding of M.nipponense has become one of the important directions of aquaculture development in the Yangtze River Delta region.Rational and effective utilization of salt-alkali water resources can expand the aquaculture space,and provide a new way for the sustainable development of our fishery.M.nipponense is one of the economic shrimp species suitable for salt-alkali aquaculture in China.The comprehensive utilization of salt-alkali water for artificial aquaculture of M.nipponense and the development of salt-alkali fishery provide the possibility for the comprehensive ecological management of salt-alkali land region.Salinity stress has become an urgent environmental problem to be solved in salt-alkali fishery which affects physiological and biochemical characteristics of M.nipponense.M.nipponense adapts to environmental salinity changes through the pathways of osmotic pressure and acid-base balance regulation.However,the molecular regulatory mechanisms of M.nipponense in response to salinity stress has not been studied systematically and thoroughly.In this study,omics technology was used to preliminarily explore the effects on transcriptomes and proteomes,as well as related genes and proteins of M.nipponense under high salinity stress.The key regulatory pathways such as energy metabolism,osmotic regulation,ion transport,immune stress,amino acid metabolism and antioxidant system were comprehensively analyzed.Based on omics analysis,the GLUT1 and GLUT2 genes of glucose transporters were excavated,and the full-length c DNA of GLUT1 and GLUT2 gene was cloned by c DNA terminal rapid amplification(RACE)PCR technique,and the gene expression was analyzed under high salinity stress.The contents and main results of this study are as follows:1.Comparative transcriptomic analysis of gill and hepatopancrea tissues of Macrobrachium nipponense under acute high salinity stressIn order to investigate the effects of salinity acclimation on the transcriptome of gill and hepatopancreas tissues,as well as the expression levels of key genes related to salinity stress of M.nipponense,360 healthy shrimps with body weight of(2.15±0.20)g were selected and were domesticated in freshwater(salinity～0),low salinity water(salinity 6±0.2)and high salinity water(salinity 12±0.2).Transcriptome sequencing and analysis were conducted in gill and hepatopancreas tissues of M.nipponense which acclimated in different salinity.The results showed that a total of 162,250 unigenes were generated by transcriptome sequencing,3,220 and 3,167 differentially expressed genes were screened from the comparation of low salinity group and freshwater control group,as well as the high salinity group and control group,respectively.2,405 and2,671 differentially expressed genes were filtered in hepatopancreas,respectively.GO and KEGG analysis showed that glycolysis/gluconeogenesis,TCA cycle,oxidative phosphorylation were significantly enriched.RT-q PCR was used to verify the expression profiles of 12 differentially expressed genes in gill and hepatopancreas tissues under acute high salinity stress,including carbonic anhydrase,Na~+/K~+-ATPase,catalase,copper/zinc superoxide dismutase,manganese superoxide dismutase,glucose transporter 1,glutathione peroxidase 3,hexokinase,L-lactate dehydrogenase,phosphofructokinase,pyruvate dehydrogenase,and heat shock protein 70.In situ hybridization localization of carbonic anhydrase gene showed that it was mainly localized in the gill columnar epithelial cells of M.nipponense.Target transcripts and key genes of salinity stress play key roles in response to salinity acclimation of M.nipponense.The results of this experiment can provide scientific basis for salt-alkaline water cultivation of M.nipponense.2.Comparative proteomic analysis of gill tissues of Macrobrachium nipponense under chronic high salinity stressTo further investigate the effects of salinity acclimation on the proteomes and related proteins expressions of M.nipponense,360 healthy shrimps with body weight of(2.15±0.20)g were selected and were domesticated in freshwater(salinity～0),low salinity water(salinity 6±0.2)and high salinity water(salinity 12±0.2).A total of 26,350 polypeptide fragments and 2,636 proteins were identified by proteome sequencing.The protein profiles of control group and the high salinity group,as well as control group and the low salinity group were compared,and 97 and 94 differentially expressed proteins were screened,respectively.GO and KEGG analysis predicted several pathways related to salinity regulation,such as glutathione metabolism,endoplasmic reticulum protein processing,serine and threonine metabolism,oxidative phosphorylation.The expression profiles of 10 significantly differentially expressed proteins in the gill tissues of M.nipponense under chronic high salinity stress were verified by RT-q PCR,including glutathione-S-transferase,heat stress protein 60,glutathione peroxidase,crustaceans calcium-binding protein,isocitrate dehydrogenase 1,manganese superoxide dismutase,peroxidase,glucose transporter 1,heat stress protein70,and glucose transporter 2.It was found that the key proteins involved in glucose metabolism,antioxidant system,heat stress response,amino acid metabolism and other pathways played the key roles in the response to chronic high salinity stress of M.nipponense.The micromethod was used to detect the enzyme activities in gill tissue under chronic high salinity stress.The results showed that,compared with the fresh water control group,the activities of catalase,glutathione-S-transferase and superoxide dismutase in the low salinity stress group were significantly increased,and the activities of acid phosphatase were decreased with the increase of salinity,while the activities of alkaline phosphatase and malondialdehyde were first increased and then decreased.In conclusion,the key proteins of salinity regulation played the key roles in response to salinity acclimation of M.nipponense,and the activities of key enzymes in the gill tissues of M.nipponense are inhibited by high salinity stress.The results of this study laid a theoretical foundation for further analysis of the molecular mechanism of response to high salinity stress,subsequent physiological researches and practical productions of M.nipponense.3.Cloning and expression analysis of glucose transporter GLUT1 and GLUT2genesThe results of transcriptomic and proteomic analysis showed that glucose transporter 1(GLUT1)and glucose transporter 2(GLUT2)were significantly differentially expressed.To explore the roles of GLUT1 and GLUT2 genes in salinity regulation pathways of M.nipponense,c DNA terminal rapid amplification(RACE)PCR was used to clone the full-length c DNA sequences of GLUT1 and GLUT2 genes,and bioinformatics analysis was performed using online softwares.RT-q PCR was used to verify the expression profiles of GLUT1 and GLUT2 genes under high salinity stress,and Western blot was used to detect the expressions of GLUT1 and GLUT2 proteins in the gill tissues of M.nipponense under chronic high salinity stress.The GLUT1 c DNA sequence was 3,020 bp(NCBI entry number:MT733824),the 5’non-coding region(UTR)was 150 bp,the 3’UTR was 1,370 bp,and the open reading frame(ORF)length was 1,497 bp,encoding 499 amino acids.The full c DNA sequence of GLUT2 gene was2,250 bp(NCBI entry number:OM049769),the 5’non-coding region(UTR)was 420bp,the 3’UTR was 402 bp,and the open reading frame(ORF)was 1,428 bp,encoding476 amino acids.Bioinformatics analysis showed that GLUT1 and GLUT2 gene had a highly conserved MFS functional domain and was highly homologous with Penaeus spectus.The results of RT-q PCR showed that compared with the control group,the expression levels of GLUT1 and GLUT2 genes increased significantly after 48h of acute high salinity stress and decreased after 14 days of chronic high salinity stress.Western blot results showed that chronic high salinity stress inhibited the expressions of GLUT1and GLUT2 proteins in the gill tissues of M.nipponense.In conclusion,GLUT1 and GLUT2 genes in gill tissues are involved in the molecular regulation process to response the salinity stress of M.nipponense.This study can provide theoretical references for the further analysis of regulatory mechanisms of M.nipponense in response to changes in osmotic pressure.