MRNA Expression And Prokaryotic Expression Study Of Goose FSH-α Gene

The low efficiency of reproductive capacity is the key problem in goose production industry, as the ovulatory cycle of goose is a long time which has clearance period as well. Follicle-stimulating hormone (FSH) is the main candidate gene that regulating the growth of avian follicle. More and more evidences demonstrated that FSH induced the synthesis of pregnendione by second mature follicles, playing a role on the small follicles which inducing the synthesis of estrogen, and then promoting the maturation of follicle. According to the change rules of expression of FSH gene and its negative regulator INH gene in goose reproduction cycle, we revealed the regulate function of FSH in the process of goose egg laying in this research. Otherwise, in order to develop biologics of goose rFSH and increase the egg production of goose, we inserted the CTP sequence which could prolong the half-life of gene into the end of each FSH subunit sequence by fusion PCR, and constructed fusion proteins, such as pET32a-FSH α-CTP, pET32a-FSH β-CTP and pET32a-FSH β-CTP-α.We determined the expression level of FSH-α and INH-α genes in different goose tissues at different reproductive period by both RT-PCR and RT-qPCR. The results demonstrated expression of FSH-α and INH-α are both expressed in heart, liver, spleen, hypothalamus, kidney, large intestine, small intestine, brain, cerebellum, fallopian tube, glandular stomach, hypophysis, lungs, ovary and pectorales etc, and especially high in reproductive tissues such as hypophysis and ovary. We also found the expression level of FSH-α gene in egg laying period (before ovulation) is significantly higher than other three periods (periparturient period, egg laying period (after ovulation) and hatch period). But the expression level of INH-α gene in egg laying period (before ovulation) is the lowest, which is very different from FSH-α. The result revealed the change rules of FSH-α gene and INH-α gene at different reproduction periods in goose.According to the sequence information of goose FSH gene published on NCBI and the character of pET32a expression vector, we designed specificity primers with restriction enzyme sites but without signal peptides, then we acquired target genes FSH α-CTP, FSH β-CTP and FSH β-CTP-α by fusion PCR and constructed expression vectors pET32a-FSH α-CTP, pET32a-FSH β-CTP and pET32a-FSH β-CTP-α and transferred them into E.COLI BL21(DE3). Proteins were purified after inducing by IPTG and checking of SDS-PAGE. The results confirmed that the best inducing time of IPTG of three fusion proteins FSH α-CTP、FSH β-CTP和FSH β-CTP-α are4h,5h and5h, respectively, and the best inducing concentrations of IPTG are respectively0.2mmol/L,0.8mmol/L and1.5mmol/L, all fusion proteins are existed in the form of inclusion body. As expected, the molecular weight of FSH α-CTP, FSH β-CTP and FSH β-CTP-α is32KD,35KD and46KD respectively. Our data lay the foundation for immunity study of goose FSH gene and development on biologics of goose rFSH.