Transcriptomics Of Gymncypris Eckloni Under Hypoxia And The Expression Regulation Of Major Differentially Genes

Gymncypris eckloni is one of the representative species from subfamily Schizothoracinae,which is the largest and most diverse taxon of the Qinghai-Tibetan Plateau ichthyofauna.In view of the importance of G.eckloni on the plateau freshwater ecosystem and its well ability of adaptation to plateau hypoxia,we sequenced the transcriptome of mixed tissues including hepatopancreas,muscle,brain,kidney and blood from G.eckloni by HiSeqTM 2500 technology.We applied normoxic(DO = 8.4 ± 0.1 mg/L)and hypoxic(DO = 3.0 ± 0.1 mg/L)treatments to analyze differentially expressed genes and their pathways related to hypoxia adaptation.Finally,we used Real-time PCR to detect the mRNAexpression changes of major genes related to hypoxia adaptation in G.eckloni under severe hypoxia and moderate hypoxia.The aim of the study was to elucidate the biological processes and molecular mechanism of hypoxia adaptation in G.eckloni.The main findings are as follows:(1)De novo assembly analysis based on all Illumina reads identified a total of 551 430 transcripts,with the length of 476 992 977 bp.Further assembly analysis showed that all transcripts contributed to 337 481 unigenes,and the size was 221 260 249 bp.De novo transcriptome assembly of G.eckloni met to the basic requirements for subsequent transcriptomic analyses.(2)The unigenes were subjected to annotation analysis by matching sequences against Nr,Nt,Pfam,KOG/COG,Swiss-prot,KEGG,and GO databases.the results showed that the Nt database accounted for the annotations(117 993,34.96%),followed by GO database(50492,14.96%),the smallest was the K EGG database(20 660,6.12%).According to the Nr database annotations,62.1% of unigene have the genetic similarity with zebrafish,which is consistent with the classification status that the two species belong to the Cyprinidae.(3)The analysis of differently expressed genes in the tissues of G.eckloni under hypoxia showed that there were 28,30,74,60 and 282 differently expressed genes in liver,heart,blood,brain and muscle tissues,respectively.Only three tissues showed GO classifications of the differently expressed genes.The genes mainly enriched in the brain and liver were related to molecular function;and the genes enriched in muscle were assigned to molecular function,cellular component,and biological process.(4)Enrichment by KEGG pathways indicated that 11 differentially expressed genes in blood were assigned to nine KEGG pathways;Five differentially expressed genes in brain tissue were assigned to five KEGG pathways;75 differentially expressed genes in muscle tissue were assigned to 29 KEGG pathways;13 differentially expressed genes in liver were assigned to 10 KEGG pathways.In response to hypoxia,fish enhanced glycolysis and gluconeogenesis,strengthened the function of HIF-1 signaling pathway and antioxidant defense system,suppressed cell growth and proliferation,and promoted apoptosis of damaged cells,thus improved hypoxia tolerance and survival ability.(5)Analysis of HIF mRNA expression revealed that the mRNA expression changesof HIF-1a A/B and HIF-2?A/B under severe and moderate hypoxia showed tissue-specific,and depended on the hypoxia regime,suggesting there may be two different mechanisms for G.eckloni to adapt to chronic hypoxia(moderate hypoxia)and acute hypoxia(severe hypoxia).(6)Under severe hypoxia,EPO mRNA expression was increased significantly in brain and muscle tissues;While under moderate hypoxia,EPO mRNA expression showed a short-timeincrease and then decreased in major tissues.This result suggested that G.eckloni enhances the oxygen carrying capacity of blood by enhancing the expression of EPO to improve hypoxia tolerance At the same time,the red blood cells are kept at normal level by the EPO feedback mechanism,and maintain the normal physiological function of the body under a long-term hypoxia.(7)The results of VEGF mRNA expression under severe and moderate hypoxia indicated that G.eckloni was able to adapt to the short-term hypoxic environment by enhancing the expression of VEGF mRNA in severe hypoxia by promoting angiogenesis and increasing blood vessel permeability.The negative feedback mechanism of the VEGF mRNA system may regulate the formation of new blood vessels to adapt to long-term hypoxia.