Screening And Functional Studies Of Genes Related To Hypoxia Stress In Macrobrachium Nipponense Based On Omics

Macrobrachium nipponense,also known as oriental river shrimp,is widely distributed in freshwater waters all over China.Because of its delicious taste and rich nutrition,especially in trace elements,M.nipponense is very popular in Jiangsu and Zhejiang province.Compared with other species,the hypoxia tolerance of M.nipponense is weak.In the actual production,the high temperature and low pressure will aggravate hypoxia in the aquaculture water,causing the phenomenon of M.nipponense,such as weakening their activities,stopping feeding and jumping out of the water.If the hypoxic environment cannot be improved in time,it will eventually cause large-scale death,which will greatly affect the increase of product income and reduce the efficiency of aquaculture.Therefore,the systematic screening and functional research of genes related hypoxia stress in M.nipponense can not only help clarify the response mechanism of M.nipponense under hypoxia stress,but also can guide the breeding of new hypoxic resistant species.This study intended to take M.nipponense as the research object,and systematically screen the hypoxia stress related genes of M.nipponense by collecting the hepatopancreas of M.nipponense in different survival states(survived,moribund and dead)under hypoxic conditions.The important differentially expressed genes for further study were screened by means of independent transcriptomic analysis and combined metabolomic analysis.The screened important differentially expressed genes were cloned and analyzed for temporal and spatial expression patterns.And their functional role in the mechanism of hypoxic stress of M.nipponense was studied by means of tissue localization.The relationship between the hypoxia related genes were confirmed by RNA interference.1 Transcriptome analysis of hepatopancreas from different living states M.nipponense in response to hypoxiaThrough the previous experiment,the appropriate dissolved oxygen concentration was selected for hypoxia stress experiment.The hepatopancreatic tissues of M.nipponense in the three survival states(survived,moribund and dead)under hypoxia were collected and compared with those in the control group for transcriptome analysis.A total of 94.22 Gb clean reads were obtained and assembled into 54,688 unigenes.A total of 54,688 unigenes were identified by function annotation.18,692(34.18%),8,178(14.95%),10,585(19.36%),12,466(22.79%),and 9,527(17.42%),genes were found to be homologous to the sequences in the Nr,Gene Ontology(GO),Swiss-Prot,KOG,and KEGG databases,respectively.A total of 19,704 unigenes were matched with at least one database.A total of 224,266,and 750 differently expressed genes were found in the comparison of the control and death groups,the control and moribund groups,and the control and survived groups,respectively.Three signal pathways closely related to hypoxia were found by enriching of the signal pathways in three comparison groups.In addition,much attention was focused on the differential genes in these pathways.Oxidative stress related genes,such as 70 k Da heat shock protein(Hsp70),phosphoenolpyruvate carboxykinase(PEPCK)and cyclooxygenase(COX)were differentially expressed in different comparisons.After comparing with previous studies,COX was found to be an important gene related hypoxia stress,meanwhile it is fully involved in the hypoxic response.Interestingly,two new genes with no Nr annotation were found in this study.2 Integrated metabolomics and transcriptomic analysis of hepatopancreas in different living status M.nipponense in response to hypoxiaWhen the study of genes related to hypoxia stress enters the transcriptome era,these hypoxia study based on transcriptome has brought a huge amount of available information,including proving the accuracy of previous homologous cloned genes are indeed related to hypoxia and a large number of unknown hypoxia related genes.However,huge of data not only bring the hope to reveal the molecular mechanism of hypoxia,but also increase the difficulty of screening key genes.Now it has become a new research trend to screen key genes through the combined analysis of transcriptomics and metabolomics.A total of 108,86 and 48 differentially expressed metabolites(DEMs)were found in three different comparisons,respectively.32 kinds of common DEMs were found by comparing different living status M.nipponense with the control group in response to hypoxia.16 genes closely related to hypoxia in M.nipponense were found by screening of common different metabolites in the three comparisons and combined with the existence of differential genes.In addition to the widely known related to hypoxia,such as PEPCK,hexokinase(HK),L-lactate dehydrogenase(LDH)and triosephosphate isomerase(TPI)were found in this manuscript.The genes were not proved to be related to hypoxia in crustacean before,such as Wnt5,delta-1-pyrroline-5-carboxylate synthase-like(P5CS),enolase(ENO)and arginase(ARG)were first time to be found in M.nipponense.In addition,PEPCK-2 was the only one differentially expressed gene(DEG)ranking high in transcriptome analysis alone and combined with metabolome analysis.The behavior of hypoxia related genes inferred from metabolites fills the one sidedness of previous hypoxia transcriptome analysis.3 Molecular cloning,expression and function analysis of key genes related to hypoxia stress in M.nipponenseThe results of transcriptomics and metabolomics showed that glutathione-Stransferase(GST)and glutathione peroxidase(GPx)not only exist in the omics joint analysis library library of different living status M.nipponense,but also play an important role in the study of homologous cloning of genes related to hypoxia stress.These two genes had two gene subtypes,respectively.These two genes were selected for follow-up research,It not only helps to promote the research of glutathione metabolic pathway and the construction the molecular mechanism of hypoxia response,but also helps to promote the study of functional differences in gene families.Herein,to elucidate thetissue-specific expression pattern of GSTs in response to hypoxia stress,which induces cell death,we investigated the expression of GSTs in response to hypoxia and reoxygenation(oxygen recovery)in M.nipponense.Full-length c DNAs of two δ class GSTs were cloned from thehepatopancreas,and named Mn GST-1 and Mn GST-2 based on the established GST nomenclaturesystem.Quantitative realtime reverse transcription PCR(q RT-PCR)was used to quantify Mn GST-1 and Mn GST-2 m RNA expression levels in different tissues,and both Mn GST-1 and Mn GST-2 were detected in all tissues tested.Mn GST-1 m RNA levels were high in the hepatopancreas,gill,muscle,ovary,and abdominal ganglion.High-level expression of Mn GST-2 was observed in the eye,brain,heart,hepatopancreas,gill,muscle and ovary.Following exposure to hypoxia,expression of Mn GST-1 and Mn GST-2 in the hepatopancreas was minimal after 12 h,but increased thereafter.The expression levels in the hypoxia group were no significantly different from those in the normal group at0 h and after reoxygenation for 12 h(p < 0.05).This trend was the same for Mn GST-2.Under reoxygenation conditions,Mn GST-1 expression returned to normal levels,but this was not the case for Mn GST-2.In the gill,Mn GST-1 expression was decreased significantly in the hypoxia group at 12 h.In muscle,expression of Mn GST-1 peaked at 12 h,and the situation was similar for Mn GST-2,although the peak was reached after reoxygenation for 24 h.In the chronic hypoxia experiment,the gene expression of Mn GST-1 in hepatopancreas and gill tissue had the same trend.After 28 days,there was no significant difference between the two hypoxia groups,but it had significant difference between the hypoxia group and the control group(p < 0.05).But this trend did not appear in muscles.For Mn GST-2,the expression pattern in hepatopancreas and gill tissue is similar to that of Mn GST-1.The difference was that Mn GST-1 in the control group is significantly higher than that in the two hypoxic groups,but the expression of Mn GST-2 in the control group was significantly lower than that in the two experimental groups.According to the glutathione peroxidase naming system,the two subtypes of GPx found in omics joint analysis library of different living status M.nipponense under hypoxia were named Mn GPx-3 and Mn GPx-4 respectively.The full-length c DNA of both subtypes were cloned from hepatopancreas.The m RNA expression of two Mn GPx genes in hepatopancreas under different oxygen environment were detected by q RTPCR.When exposed to hypoxia,Mn GPx-3 reached its maximum at the 12 th hour of hypoxia,but then decreased.The expression of Mn GPx-4 in hepatopancreas reached the peak at 12 hours of reoxygenation.Under the condition of reoxygenation,the expression of Mn GPx-4 returned to the normal level,but Mn GPx-3 did not.The expression of Mn GPx-3 in gill tissue was similar to that in hepatopancreas,but the difference was that the expression of Mn GPx-3 in gill tissue returned to normal after24 hours of reoxygenation,but the expression of Mn GPx-3 in gill tissue decreased continuously after reoxygenation.In situ hybridization showed that Mn GPx-3 and Mn GPx-4 were located in secretory and storage cells in hepatopancreas.These results suggest that GPx gene is expressed and released by secretory cells and released response to hypoxia.In the gill tissue,however,GPxs were located in blood cells,suggesting that they perform different functions in different tissues or organs.The results of in situ hybridization were consistent with those of q RT-PCR.This study provided a basis for understanding the oxidative stress response in M.nipponense under hypoxia.4 Mutual regulation of key genes related to hypoxia stress in M.nipponenseRNA interference was used to inhibit the expression of HIF-1α,Mn GST-1 and Mn GST-2 genes.The regulatory relationship between genes screened by combined analysis of transcriptome and metabolome and these three genes was studied at the m RNA level.The interference results showed that the ds RNA of the three genes could significantly reduce the expression of the target gene.The results of mutual regulation showed that HIF-1α could not directly regulate the expression of HIF-1β,Mn GST-2and Mn GPx-4.HIF-1α can positively regulate the expression of Mn GST-1,PEPCK-1,PEPCK-2,PEPCK-3 and PEPCK-4.In addition,HIF-1α has a negative regulatory relationship with Mn GPx-3 and COX genes.When Mn GST-1 gene was interfered,HIF-1α,HIF-1β,Mn GPx-3,PEPCK-2,PEPCK-3 and PEPCK-4 genes showed a significant upward trend.In the experiment of Mn GST-2 gene,we can obviously find the negative regulation effect of Mn GST-2 on HIF-1β and Mn GPx-3 and its positive regulation effect on PEPCK-1 and PEPCK-4.The upstream and downstream regulation relationship between Mn GST-1 and Mn GST-2 not only indicates that different subtypes have different response mechanisms,but also indicates that there may be specific regulation between gene subtypes in crustaceans.Based on the interference results,we speculated that when M.nipponense coped with hypoxic environment,(1)Mn GST-1 gene will induced by HIF-1α,glutathione reaction will be accelerated to reduce the damage by ROS.(2)Meanwhile,HIF-1α also induced PEPCK expression and accelerated gluconeogenesis.(3)HIF-1α can inhibit the expression of the COX gene to reduce inflammation.