Gene Expression And Analysis Of GS And Akt2 In Rana Dybowskii Under Low Temperature Stress

Rana dybowskii is mainly distributed in the northeast region of China,and the area is in the sub-frigid zone.The six-month hibernation period poses a great threat to the survival of Rana dybowskii.Preliminary research of our laboratory showed that Rana dybowskii could accumulate large amounts of glucose as cryoprotectant in low temperature condition.GS(Glycogen synthase)is the major rate-limiting enzyme for glycogen synthesis,involved in the synthesis and decomposition of glycogen,thus playing an important role in glucose metabolism.Akt(Serine/threonine kinase),as an important gene in the insulin signaling pathway,is not only involved in the regulation of glucose metabolism,but also in inhibiting apoptosis and promoting cell survival under adverse stimuli.This research explored how Rana dybowskii generated such high glucose level at low temperature,laying the foundation for the decryption of high sugar tolerant mechanism.In this experiment,Rana dybowskii in the northeastern region was used as the research object and treated with low temperature.The experimental group was-1?,and the control group was 4?.The relative expression levels of GS and Akt2 were detected in the heart,liver and skeletal muscle at 4h,8h,12 h,24h,48 h and 72 h after cold stress.The GS and Akt2 gene sequences of Rana dybowskii were cloned by polymerase chain reaction(PCR)and RACE method.The relative expressions of GS and Akt2 were detected by qPCR.Experimental results: The total length of GS gene is 2855 bp,and the open reading frame is 2214 bp,encoding 737 amino acids.The total length of Akt2 gene is 1976 bp,and the open reading frame is 1461 bp,encoding 486 amino acids.The relative expression of GS in heart,liver and skeletal muscle of Rana dybowskii significantly declined under low temperature stress.In heart,the relative expression of GS reached the lowest level at 8h,and was 9% of the control group,then returned to normal at 24 h.In the liver and skeletal muscle,the relative expression of GS has been remaining at a low level,with the lowest at 4h.The relative expression of GS in liver was 17% of the control group,and in skeletal muscle,it was 4% of the control group.The relative expression level of Akt2 in heart and liver increased significantly under low temperature stress and reached the highest peak at 12 h.The relative expression of Akt2 in heart was 4.4 times of the control and 4.6 times in the liver,then returned to the initial level at 72 h.However,the change of expression in skeletal muscle was not significant.In this study,the full-length sequences of GS and Akt2 genes of Rana dybowskii were obtained for the first time,and analyzed the express of GS and Akt2 in Rana dybowskii under low temperature stress.The results showed that GS mRNA expression was down-regulated and Akt2 mRNA expression was significantly up-regulated,which providing theoretical data for further exploring the related research in hypoglycemic metabolism of Rana dybowskii.