Expression Of Lcleptin-a And Lcleptin-b Genes And Preliminary Analysis Of Their Promoters In Larimichthys Crocea

Leptin is a member of type I cytokines which is widely distributed in the body of mammals.It is involved in energy metabolism,neuroendocrine,immune response,reproduction and growth.Mammals and amphibians generally contain only one type of leptin gene,but many kinds of fish contain both leptin-a and leptin-b genes.Existing studies have shown that Leptin plays an important role in the growth and regulation of energy metabolism in fish.In this study,the realtime fluorescent quantitative PCR(qRT-PCR)technology was used to detect the expression of Lcleptin-a and Lcleptin-b genes in different tissues of large yellow croaker(Larimichthys crocea)which is an important commercially marine fish in China,and to study the effects of fasting and low-salinity acclimation on the expression of Lcleptin-a and Lclcleptin-b genes to know the biological function of Leptin in L.crocea.Meantime,the promoter region sequences of Lcleptina and Lclcleptin-b gene were obtained by the genome date of L.crocea from NCBI and were verified by PCR method.The preliminary functional analysis of the promoter region of Lcleptina was carried out by constructing the recombinant plasmid,transient transfection and dual luciferase reporter gene assay.The main results are as follows.1)The liver,hypothalamus,pituitary,muscle,kidney,gill,intestine,spleen,head kidney and skin were chosen to detect gene expression by qRT-PCR.The result showed that Lcleptin-a gene was expressed highest in liver,next in hypothalamus.Lcleptin-b gene was expressed highest in hypothalamus and pituitary,next in gonad.2)The effect of fasting on the expression level of Lcleptin-a and Lcleptin-b in the liver and hypothalamus of large yellow croaker was determined by using qRT-PCR.The result showed that a-week fasting can significantly reduce the expression of Lcleptin-a in liver and the expression of Lcleptin-b in hypothalamus,but neither of them further deduced after 2-week fasting.A-week refeeding can reduce the expression of Lcleptin-a in liver and the expression of Lcleptin-b in hypothalamus to the control group level.3)During low-salinity acclimation,the expression of Lcleptin-a gene in liver was increased significantly after the fish had been treated with 16‰ salinity water for 24h;when the salinity was diluted to 8‰ with fresh water,the expression of Lcleptin-a gene in liver was not significantly different from control group,and the expression of Lcleptin-a gene in liver was no longer decreased after treatment with lower salinity water.After treated the fish at 8‰ for 24h,the expression of Lcleptin-a in hypothalamus was significantly up-regulated(P<0.01).The expression of Lcleptin-b gene in the hypothalamus of fish that have been treated in 16 ‰salinity of the water for 24h was reduced significantly.However,after the fish had been treated in 4 ‰ salinity water for 24h,its expression was increased then significantly reduced again after the salinity was diluted to 2 ‰ and the fish had been kept in 2‰ salinity for 24 h.However after treated the fish with fresh water for 24h,its expression increased again.Different salinity may affect the expression of Lcleptin-b in liver,however,the expression level is too low to detect thus it is impossible to elucidate the role of Lcleptin-b in the liver in osmotic regulation.4)The fragments of 5’ flanking region sequences of Lcleptin-a and Lcleptin-b genes were obtained which the length is 2435 bp and 2884 bp respectively.The multiple suspected core promoters were predicted by bioinformatics software.The core promoter of Lcleptin-a located at the position of 662 bp in the 5’ flanking region of ATG upstream was verified by dual luciferase reporter gene assay.The CpG island was predicted for the promoter region of Lcleptin-a and Lcleptin-b,but neither of them contained a CpG island.5)Eight different lengths of Lcleptin-a promoter deletion fragments were constructed successfully.The result showed that the luciferase activities of pGL3-lepa-1.1 and pGL3-lepa1 were the highest but no significant difference.The luciferase activities between pGL3-lepa1 and pGL3-lepa-2,pGL3-lepa-2 and pGL3-lepa-3,pGL3-lepa-4 and pGL3-lepa-5,and pGL3-lepa-5 and pGL3-lepa-6 were significantly different.