Study Of TGF-?1 Gene Of Plateau Schizothoracine Fish In Hypoxia Adaption

The Qinghai-Tibet Plateau is located in the southwest of China(average over 4000 metres above the sea level).As a result of high altitude,there are the characteristics of small oxygen content,strong ultraviolet radiation,less rainfall and low temperature in Qinghai-Tibet Plateau.Hypoxia is one of many stressors for organisms.When oxygen is not enough,organisms are easy to be damaged,even to be death.In order to adapt to the hypoxic environment,unique physiological mechanisms have be evolved,such as the cardiovascular system,respiratory system and other systems,which have the function of stabilizing the level of oxygen in the organisms.These characteristics of plateau organisms continue to be inherited and evolved,so that they can further adapt to the plateau hypoxia environment.Hypoxic adaptation is not only manifested in organ tissue level,but also at cell level and molecular level for plateau organisms.TGF-?(Transforming growth factor-?)is a pleiotropic cytokine and play an important role in cell growth,differentiation,death as well as cellular immune response.In the previous experiment,the expression of TGF-?1 gene was increased from transcriptome analysis of Gymnocypris dobula.And it may be a sign of functional evolution of TGF-?1 in plateau schizothoracine fish.To explore the adaptation mechanism of TGF-beta 1 gene to hypoxia environment,we compared the expression in different tissues between G.dobula TGF-?1 and S.Prenanti firstly,and built the TGF-?1 eukaryotic expression vectors to make the hypoxia experiment in vivo secondly.At the same time we got the cell relative viability and protein specificity expressions.Our studies demonstrated that cell survival rate were elevated in a time-dependent manner under 1% oxygen hypoxia,the cell viability increased significant under 0.1%hypoxia stress first and then recovered to the normal levels.The oxidative stress response and physiological methbolism related indices of ZF-4 cells under different hypoxic stress were measured.Compared with control group,the activity of superoxide dismutase(SOD)were significantly higher,reaching the highest value at24 th hour,the activity of glutathione reductase(GSH)was higher and the activity of catalase(CAT)was lower than those in control at 8th hour,the content of nitric oxide and activity of nitric oxide synthase(NOS)increased significantly than that in the control group at 16 th hour,but the content of malondialdehyde(MDA)was unchanged under hypoxic with 1% oxygen;SOD and GSH activities were increased under 0.1% hypoxic treatment,the CAT activity,the nitric oxide synthase activity and the contents of MDA and NO were reduced under 0.1% hypoxic stress;Under acute hypoxia condition,the related enzyme activities and substance were changed dynamically of the ZF-4 cells.These results reveal that the activity of antioxidases were upregulated through improve the antioxidant abilities of ZF-4 cells.And ZF-4cells were dealt with hypoxia for different hours,and TGF-?1 m RNA expression levels were analyzed by q RT-PCR.The results showed that HIF,EPO and TGF-?1m RNA expression levels under 1% and 0.1% hypoxia stress were significantly increased.This showed that HIF,EPO and TGF-?1genes may play an important role in hypoxia adaptation.HIF,EPO genes have been proved,but the adaptive evolution of fish's TGF-?1 genes and how to regulate genes expression are still not completely understood under hypoxic conditions.The ORF of TGF-?1 in G.dobula and S.prenanti were1134 bp,and maybe they all encoded 377 amino acids.Amino acids alignment shows the homology between G.dobula and S.prenanti TGF-?1 as 89.66%.Compared with the zebrafish,S.prenanti shared 89.66% identities.Under the long-term hypoxia condition,the TGF-?1 m RNA expression levels of spleen,brain,heart,muscle,skin,and gill were compared in G.dobula and S.prenanti,respectively.The q RT-PCR showed that the expression levels of TGF-?1 m RNA in teleost were significantly decreased in long-term hypoxia condition.At the same time,the protein expression of active TGF-?1 in heart of G.dobula was much more than S.prenanti.Furthermore,in the hypoxia experiment in vitro,the relative viability of GD-TGF?1 transfected cells was higher than SP-TGF?1 transfected cells.It shows that G.dobula TGF-?1 can better adapt to the plateau low oxygen environment,and it has the nerve protection and issue protection function.This study carried on the molecular and protein level of TGF-?1,and provide theoretical basis to reveal the genetic characteristics of TGF-?1 gene in extreme circumstances.Furthermore,it laid the foundation of adaptive evolution of the TGF-?1 gene in fishes.