Salt-and Drought-resistance Mechanisms Of Camel Revealed By RNA-seq In Kidney,Hepar And Ileum

Camels are large mammals that live in desert for a long history.As excellent biological resources for animal resistance-related research,camels have extremely important value for sciences and applications.In this study,the transcriptome of 9 female Bactrian Camels(Camelus bactrianus)was sequenced and then analysed.The resistance genes screening,analysis of post-transcriptional regulation and verifying test were subsequently performed.We hope to provide molecular theoretical basis for revealing the unique mechanisms of the camel adapting to desert environment and solving human diseases,such as hypertension and hyperglycaemia.The 9 female Bactrian camels were randomly divided into salt stress experimental group,water-deprivation stress experimental group and free diet control group.Each group has 3 camels.After 24 days of experimental treatment,the renal cortex,renal medulla,hepar and ileum of the 9 camels were collected.Then,we used Illumina HiSeq 4000 and 2500 platform to sequence the transcriptome of the collected tissue samples.Differentially expressed messenger RNAs(mRNAs)and non-coding RNAs were screened from the clean data after quality control.Compared with the control group,13 mRNAs,4 lncRNAs and 11 miRNAs were expressed significantly differentially in the renal cortex of salt stress group,14 mRNAs,0 lncRNA and 18 miRNAs were expressed significantly differentially in the renal cortex of water-deprivation stress group,22 mRNAs,2 lncRNAs and 31 miRNAs were expressed significantly differentially in the renal medulla of salt stress group,17 mRNAs,0 lncRNA and 26 miRNAs were expressed significantly differentially in the renal medulla of water-deprivation stress group,14 mRNAs,5 lncRNAs and 12 miRNAs were expressed significantly differentially in the hepar of salt stress group 3 16 mRNAs,1 lncRNA and 37 miRNAs were expressed significantly differentially in the hepar of water-deprivation stress group,22 mRNAs,3 lncRNAs and 56 miRNAs were expressed significantly differentially in the ileum of salt stress group,and 43 mRNAs,7 lncRNAs and 36 miRNAs were expressed significantly differentially in the ileum of water-deprivation stress group.Combined with the GO and KEGG pathway analysis of the differentially expressed genes and previous researches,we selected salt-,glucose-,water-metabolism related genes as candidate genes.The candidate genes included 6 genes(SLC6A1,SLC14A1,PCBP2,PEX5L,LNC003834 and miRNA-34a)in renal medulla under salt stress,4 genes(ACLY,PCBP2,LOC105061856 and miRNA-195)in renal medulla under water-deprivation stress,6 genes(TENM1,PKP4,CDH11,SDS,LOC105061856 and miRNA-195)in hepar under salt stress,7 genes(PLIN2,SDS,UPP2,LOC105061856,miRNA-365-3p,miRNA-128 and miRNA-148a)in hepar under water-deprivation stress,2 genes(AQP5 and MUC6)in ileum under salt stress,and 6 genes(AQP5,MUC6,LOC105076960,miRNA-362-5p,miRNA-96 and miRNA-29b)in ileum under water-deprivation stress.According to the qRT-PCR assay of all candidate genes in renal medulla,hepar and ileum under salt stress and water-deprivation stress,the quantitative results of differential expressions of genes were consistent with that of sequencing results.In renal medulla under salt stress,FISH and Luciferase assay of candidate ceRNA genes(SLC14A1,LNC003834 and miRNA-34a)indicated that non-coding LNC003834 and miRNA-34a were ectopically expressed with the nucleus.miRNA-34a could specifically inhibit the mRNA of SLC14A1,while LNC003834 could reduce the inhibition by competitive binding miRNA-34a.RNAi and ROS assay of candidate antioxidant genes(SLC6A1,PCBP2 and PEX5L)demonstrated that the expression of ROS increased in the renal medulla cells that shRNA lentiviruses successful interfered with SLC6A1,PCBP2,and PEX5L,compared with that in normal renal medulla cells of camel.In summary,the regulatory model of candidate genes in the renal medulla,hepar and ileum of the camel under salt stress and water-deprivation stress was further established.In renal medulla,the gene expression of urea transporter B was up-regulated by ceRNA mechanism to reduce the amount of Na+ that reabsorbed into the blood,and the antioxidant ability of cells was also improved via up-regulating the gene expressions of GABA transporter 1,peroxisome biogenesis factor 5-related protein and poly(rC)-binding protein 2,thus realizing the salt resistance.The drought resistance of renal medulla was achieved by the decrease of cellular respiration and metabolism through down-regulating the gene expressions of ATP-citrate synthase and hemoglobin subunit p,and the improvement of the antioxidant ability of cells through up-regulating gene expression of poly(rC)-binding protein 2 in the meantime.The salt resistance of hepar was fulfilled by enhancing the effect of the permeation barrier in the cell basement membrane via up-regulating Teneurin transmembrane protein 1 gene expression,by improving cell adhesion and reducing the needed superficial area of Na+ entering into the cell via up-regulating the gene expressions of cadherin-11 and plakophilin-4,by reducing cellular respiration and metabolism via down-regulating hemoglobin subunit ? gene expression,and by decreasing gluconeogenesis and preventing intracellular sugar deposition via down-regulating the gene expression of serine dehydratase.To obtain the ability of glucose and drought resistance in hepar,gene expressions of uridine phosphorylase 2 and serine dehydratase were down-regulated to diminish gluconeogenesis and glycogen synthesis,perilipin-2 expression was up-regulated to promote lipid droplet accumulation and the secretion of milk lipid globule-like structure,and hemoglobin subunit ? gene expression was down-regulated to reduce cellular respiration and metabolism.In ileum,the salt resistance was realized by the prevention of the water loss due to the excessive extracellular osmotic pressure through down-regulating the gene expression of aquaporin-5,and by the decrease of the mucin secretion via the Na+/Ca2+exchanger and the prevention of a large amount of Na+ into cells through down-regulating the gene expression of mucin-6.Glucose and drought resistance of ileum were achieved by the down-regulated expression of aquaporin-5 gene to prevent the intracellular water loss,the down-regulated gene expression of mucin-6 to reduce the mucin secretion by Na+/Ca2+ exchanger and prevent loss of Ca2+ which can promote expression and activity of fatty acid synthase(FAS),and the gene expression upregulation of mitochondrial creatine kinase U-type to form an energy buffer,thus retarding glucose load of cell caused by the sodium-dependent glucose cotransporter.