Cloning And Expression Analysis During Different Stages Of The Reproductive Cycle Of Kiss1/kiss1R And Brain-pituitary-ovary Axis Related Genes From Chinese Alligator (Alligator Sinensis)

Kisspeptin plays an important role in the regulation of vertebrate reproduction,via combining kiss1 R,which is co-expressed in the gonadotropin-releasing hormone(GnRH)neurons.Chinese alligator(Alligator sinensis)is an endemic reptile species in China,and very little study was conducted about the mechanism of reproductive regulation on this species.In this paper,research on the expression and function of the kiss1/kiss1 R system and the hormones that are related to the brain-pituitary-gonadal(BPG)axis in the Chinese alligator through the rapid amplification of cDNA ends,immunohistochemistry,real-time quantitative PCR and dual-luciferase reporter assay system methods,that is useful to enrich our knowledge about regulation mechanism of kiss1/kiss1 R system acting on BPG axis in reptiles.Main results are as follows: 1.Using rapid amplification of cDNA ends(RACE)method,we have homology-based cloned the full-length cDNA sequences of the kiss1,kiss1 R and GnRH1 genes from the Chinese alligator for the first time and analyzed the characteristics of the amino acid sequences that are encoded by the genes.In addition,Chinese alligator kiss1 R gene promoter sequence is also obtained using homology-based cloning method.The full-length cDNA sequences of the kiss1 and kiss1 R are also firstly cloned in reptiles.1)The full-length cDNA sequence of Chinese alligator kiss1 is 816 bp,including a partial 5' untranslated region(5' UTR;4 bp),a complete open reading frame(ORF;417 bp)and a complete 3' untranslated region(3' UTR;395 bp).The ORF encodes a precursor protein of 138 amino acids(aa)with a predicted signal peptide of 19 aa.The calculated molecular weight(MW)of the Kiss1 predicted protein is 15.535 kDa with an estimated isoelectric point(pI)of 10.33.2)The cDNA sequence of Chinese alligator kiss1 R is 2348 bp in length with a partial 5' UTR(109 bp),a complete 3' UTR(1153 bp)and a complete ORF(1086 bp).The ORF encodes a precursor protein of 361 aa.The calculated MW of the kiss1 R predicted protein is 40.126 kDa with an estimated p I of 9.51.3)The full-length of Chinese alligator GnRH1 c DNA sequence is 776 bp,containing a partial 5' UTR of 68 bp,a complete ORF of 282 bp and a complete 3' UTR of 426 bp.The ORF encodes a precursor protein of 93 aa with a predicted signal peptide of 23 aa.The calculated MW of the kiss1 R predicted protein is 10.356 kDa with an estimated p I of 6.09.4)The obtained promoter sequence of Chinese alligator kiss1 R gene is 2604 bp in length,which highly homology with other corresponding sequences of crocodilian.Furthermore,the sequence is also found 4 predicted promoter regions,TATA box,CAT box,GC box and several potential transcription factor binding sites,such as Sp1,C/EBP,YY1 and some steroid hormone receptors binding sites.The existence of these predicted binding sites may suggest that these transcription factors play an important role in regulation of the secretion and expression of kiss1 R.The bioinformatics and phylogenetic analysis have been used in these obtained amino acid sequences.Multiple sequence alignment analysis at the protein level shows that these predicted amino acid sequences share highly homology with that of other crocodilian species.These results indicate that the kiss1,kiss1 R and GnRH1 genes in crocodilian species are conserved in evolution.Additionally,the kiss1-10 of kiss1,the seven transmembrane domains of kiss1 R and the GnRH1-10 of GnRH1 are also highly conservative in vertebrates.The phylogenetic trees for aa sequences of the kiss1,kiss1 R and GnRH1 precursor proteins are constructed,which show that all crocodilian species are grouped with turtles.These results suggest that crocodilians close evolutionary relationships with turtles.2.Localization analysis of Chinese alligator kiss1 protein in various tissues and steroid hormone receptors(ESR1,ESR2,AR and PR)proteins in the brain tissues use the immunohistochemical method.The result shows that these proteins are widely expressed in various tissues and have common distribution areas in the brain tissues.This may indicate that sex steroid hormones regulate the expression of kiss1/kiss1 R system via their cognate receptors in the brain of Chinese alligator.3.The expression of kiss1,kiss1 R,GnRH1,GnRH2,ESR1,ESR2,AR and PR genes quantitatively in different tissues(spatial variation)and during the reproductive cycle(temporal changes)using quantitative real-time PCR(qPCR).The results show that all genes are widely expressed in various tissues,except GnRH2 gene is expressed only in the brain and the pituitary.The expression levels of these genes are high in the reproductionrelated tissues,such brain,pituitary,gonad and liver.The gene expression levels changed significantly during different stages of reproductive cycles in female Chinese alligator.These results may suggest that these genes are involved in the process of reproduction and development of ovary of female Chinese alligator.4.The full-length ORF sequence of Chinese alligator kiss1 R gene is synthesized using full gene synthesis method,and we successfully build the dual luciferase expression vector containing the ORF sequence of Chinese alligator kiss1 R gene.Then we use different synthetic hormones to act on the Chinese alligator kiss1 R gene transfection HEK293 cells.The transcription activity changes of Chinese alligator kiss1 R gene are detected by dual luciferase report system.The result suggests that estradiol(E2)and all of the synthetic kiss1-10 peptides significantly improve the transcriptional activity of Chinese alligator kiss1 R.And the Chinese alligator kiss1 R signal is transduced mostly via the protein kinase C(PKC)pathway.In addition,a variety of synthetic kiss1-10 peptides activated Chinese alligator Kiss1 R with different potencies,indicating a differential ligand selectivity.These date are first reported in reptiles.Determining Chinese alligator kiss1 R signal transduction pathway and ligand selectivity,which lay the foundation for the future research on kiss1/kiss1 R system of other reptiles,but also expand our understanding of the molecular coevolution of kiss1 and kiss1 R pairs.To sum up,kiss1/kiss1 R system may regulate reproduction-related activities in the brain,pituitary gland and gonad of Chinese alligator.Furthermore,the sex steroid hormones may also regulate the expression of kiss1/kiss1 R system through their cognate receptors in the brain and pituitary gland.E2 and all of the synthetic kiss1-10 peptides exert their functions mainly by activating PKC signal transduction pathway.