Molecular Cloning And Analysis Of Bioinformatics Of Gonadotropins Gene In Huso Dauricus

Kaluga(Huso dauricus),the species originated in the 130 million years ago Cretaceous,is the world's only rare waters of the Heilongjiang River.As one of the paleontology preserved from the Cretaceous period,it has lived with the dinosaurs on earth,and its original quaint shape has never changed almost 100 million years,so it has valuable scientific research value.Kaluga is now a national second-class wildlife,which has identified as an endangered species by the 1998 United Nations Convention on Washington.In addition,the sturgeon dwarf has a high economic value,its eggs are called "black gold",caviar is extremely delicious.But the growth of sturgeon husk is particularly slow,sexual maturity cycle in more than 18 years,so it is imperative to explore the breeding physiology of sturgeon.Gonadotropin(GtH)is a major endocrine factor secreted by the pituitary gland to promote gonadal development and reproduction.Including follicle-stimulating hormone(FSH),luteinizing hormone(LH),thyroid hormone(throtroid stimulating hormone,TSH)with the composition of the glycoprotein hormone family.The formation and release of gonadotropin secreted by the pituitary is directly regulated by luteinizing hormone releasing hormone(LH-RH)and follicle-stimulating hormone-releasing hormone(FSH-RH).Fish sexual maturity is directly related to the secretion of gonadotropin,so the use of biological means to accelerate the development of gonadotropin gene research is essential.In this study,we cloned the FSH? subunit and LH? subunit of the steric gonadotropin were genotyped by reverse transcription(RT-PCR)and rapid cDNA end amplification(RACE)The phylogenetic tree was constructed and the amino acid was analyzed by homology.At the same time,the expression levels of FSH? subunit and LH? subunit in ten tissues were analyzed by real-time fluorescence quantitative PCR.In addition,the open reading frame of gonadotropin was introduced into Escherichia coli and expressed in prokaryotic cells.The protein was purified and identified by western blot.The results are as follows:(1)The specific primers were designed to amplify the middle fragment,and the 3 'and 5' ends were amplified by RACE method.Finally,the full-length cDNA was clipped,596 bp of the LH? subunit cDNA,1090 bp of the FSH subunit cDNA,the ORF are 414 bp and 387 bp,encoding 137 and 129 amino acids respectively.Compared with other sturgeons,the amino acid homology was up to 98%.The secondary structure,tertiary structure,hydrophobicity and transmembrane region of the protein were analyzed,and the phylogenetic tree was constructed.The phylogenetic tree was constructed,which was close to the Chinese sturgeon and was far farther with other bony fishes,and farthest in humans and mice in mammals.(2)The quantitative analysis of 10 tissues of pituitary,brain,muscle,testis,ovary,liver,kidney,intestine,stomach and gill was performed.The results showed that the expression of FSH subunit and LH subunit was consistent in most tissues,and the expression of FSH subunit was the most significant,a little in the testis and ovary,no expression in the other organization.The difference of the two subunits was mainly manifested in the gonads.The FSH subunit was higher in the testis and the LH subunit was higher in the ovary.(3)The open reading frame of LH? subunit and FSH? subunit was introduced into Blunt-E1,and then the plasmid was transferred into BL21(DE3)to construct prokaryotic expression vector.Protein expression was induced by IPTG,and the expression conditions were optimized.The results showed that the concentration of IPTG had little effect on protein expression,and the optimal expression conditions were as follows: 37 induced 5h.?The fusion protein could be combined with the cloned antibody and the immunological activity was good,which indicated that the fusion protein could be successfully expressed in BL21(DE3).