Gene Structure Of Globin Superfamily Members And Their Role In Hypoxic Adaptation Of Schizopygopsis Pylzovi

The water environment of Tibetan Plateau has low dissolved oxygen,which fluctuates in different characteristics of time,space,and seasonal.The plateau native Schizothoracinae formed a unique mechanism to adapt to the extreme environment of plateau hypoxia stress through a long evolutionary adaptation.This study uses a new generation of sequencing technology to determine the main members of the globin family in Tibetan schizothoracine fish Schizopygopsis pylzovi,followed the full-length cDNA sequence of HbA,HbB,Cygb1,and Cygb2 gene and complete encoding sequence of Mb,Ngb gene of S.pylzovi was further cloned by RT-PCR and Smart-RACE.Using semi quantitative RT-PCR method to detect the expression of each gene in the S.pylzovi tissue.Using Real-time PCR and Western-blot to detect the mRNA transcription and protein levels of globin family in tissues of S.pylzovi after hypoxic treatment for 4H and moderate hypoxia treatment for12 h,24h,36 h,48h,60 h and 72 h.The main results are as follows:(1)There have been identified 269100 transcripts and 192934 unigenes from mixed tissue transcriptome of S.pylzovi through a new generation of high-throughput sequencing technology.The database search identified the key members of globin family of S.pylzovi including HbA,HbB,Mb,Cygb1,Cygb2 and Ngb.(2)The length of HbA cDNA in S.pylzovi was 657 bp,and the length of ORF was 432 bp,the length of 5'-UTR was 102 bp and the length of 3'-UTR was 123 bp.The ORF encoded 143 amino acids.The total length of HbB cDNA in S.pylzovi was 674 bp,the length of ORF was 447 bp,the length of 5'-UTR was 93 bp,and the length of 3'-UTR was 134 bp.The ORF codes 148 amino acids.The HbA of S.pylzovi was expressed in the heart,liver,muscle,kidney,eye,intestines,gill and brain.And HbB were expressed in other tissues except the intestinal gland.The Hb transcription activity of the S.pylzovi was decreased rapidly under severe hypoxia.Under the condition of moderate hypoxia,the HbB transcriptional activity was elevated in a short time,but the transcription mechanism was slowly decreased under prolonged exposure to moderate hypoxia.In addition,studies have shown that the kidneys are not only used as erythropoiesis,but also as the main blood bank of S.pylzovi.(3)The length of Mb encoding sequence of S.pylzovi was 444 bp,encoding 147 amino acids,and no paralogs were found in the transcriptome data of S.pylzovi displaying there is only one Mb gene in S.pylzovi;The Mb gene was expressed in heart,liver,muscle,kidney,eye,intestine,gill,brain and skin tissues of S.pylzovi.The Mb mRNA expression level in liver,muscle and brain of S.pylzovi was slightly decreased under moderate and severe hypoxia,while Mb mRNA and protein expression level was parallel increased in the heart in moderate and severe hypoxia.Severe hypoxia induced the Mb mRNA and protein significantly expressed in the heart of S.pylzovi.(4)The total length of Cygb1 cDNA was 1207 bp,the length of ORF was 534 bp,the length of 5'-UTR was 95 bp,and the length of 3'-UTR was 578 bp.The ORF encoded 177 amino acids.The total length of Cygb2 was 1121 bp,the length of ORF was 534 bp,5'-UTR was 223 bp,and the 3'-UTR was 578 bp.The ORF encoded 179 amino acids.The Cygb1 and Cygb2 genes both expressed in the liver,brain,kidney,spleen,heart,muscle,eye and gill.The expression of Cygb1 in the liver,kidney and brain was significantly lower than that in the brain,and the expression of Cygb2 in the muscle and liver was significantly decreased but in other tissues remained sTab.after severe hypoxia for 4 hours.The transcription activity of Cygb1 and Cygb2 gene in muscle and brain decreased rapidly after moderate hypoxia hypoxia.And in the liver,kidney and heart,the transcriptional activity of the two genes have a short-term uptake in different time periods exposed to hypoxia.(5)The lenth of Ngb encoding sequence is about 480 bp and encoded 159 amino acids.The Ngb gene is mainly expression in brain and eye tissues of S.pylzovi,in the liver,kidney,heart and gill tissues are still detected,but the expression level is low.The expression level of Ngb mRNA in brain and eye tissues was significantly decreased under the condition of severe hypoxia.The expression of Ngb mRNA was significantly up-regulated in the brain tissue of S.pylzovi within 12 h after moderate hypoxia,followed by a significant downward adjustment and then showed a slow rise in the volatility of the change.