Cloning,Expression Analysis Of Gth??Fshr And Kisspeptin Reproductive Genes In Amur Sturgeon(Acipenser Sturgeon)

Acipenser schrenckii,which originated in the Cretaceous period,is a typical and large freshwater economic fish with high scientific research value and economic value.As the first independently developed and important sturgeon breed in China,Shi's sturgeon has the advantages of fast growth and wide range of culture.At present,there are few reports on the theoretical basis of sex differentiation and gonad development regulation of sturgeon in the world,and its molecular regulation mechanism has not been clarified yet.The lack of effective all female breeding technology means has become a bottleneck problem in the development of sturgeon caviar production from female sturgeon eggs,which restricts the healthy and sustainable development of sturgeon industry in China.Therefore,this paper takes sturgeon schrenckii as the research object,and uses reverse transcription polymerase chain reaction(RT-PCR),rapid amplification of cDNA ends(RACE)and nucleic acid sequencing technology to regulate the development of "hypothalamus pituitary gonad" axis of sturgeon schrenckii The full cDNA sequences of Gth?,Fshr and Kiss genes were cloned and bioinformatics analyzed.At the same time,RT-q PCR was used to carry out the tissue expression characteristics and temporal and spatial changes of related genes during the early gonad development of sturgeon schrencki.The main results are as follows:1.The Gth? gene of Acipenser schrenckii has a total length of 645 bp,including 54 bp of 5?noncoding region,348 bp of open reading frame and 243 bp of 3?noncoding region.The gene encodes 115 amino acids.The secondary structure of protein is mainly ?-helix and irregular curl,which belongs to hydrophilic protein with signal peptide,transmembrane structure and secreted to the outside of cells.The analysis of amino acid homology and evolution showed that the GTH? sequences of Acipenser sinensis and Amur sturgeon were the most consistent,and they were closely related to sturgeons.The expression of Gth? gene in gonad and pituitary was higher than that in muscle and in-testine(P < 0.05).2.The total length of Fshr gene is 2696 BP,including 147 bp 5?untranslated region,1986 BP open reading frame and 608 bp(including poly A tail)3?untranslated region,encoding 661 amino acids.The analysis of amino acid homology and evolution showed that the FSHR sequence of Acipenser ruthenus and Acipenser sturgeon was the most consistent,and the genetic relationship was the closest.The results of PCR showed that the expression of Fshr m RNA was widespread in all tissues,but it was higher in gonad and pituitary than in other tissues(P<0.05).3.The results indicated that cDNA of the Kiss1 gene of Acipenser schrenckii was 522 bp,encoding 130 amino acids;cDNA of the Kiss2 gene of Acipenser schrenckii was 518 bp,encoding 149 amino acids;Among them,the ?-helix and irregular distortion in KISS1 and KISS2 are regarded as the main components in the secondary structure,both contains signal peptide,transmembrane structure,and unstable hydrophilic protein secreted outside the cell.Amino acid sequence alignment and phylogenetic tree analysis showed that KISS1 and KISS2 of Acipenser schrenckii had highly conserved regions,which had the highest consistency with the KISS1 and KISS2 sequences of Acipenser dabryanus,and the closest relationship.The results of real-time PCR showed that the expression of Kiss1 and Kiss2 in Acipenser schrenckii was tissue-specific,and Kiss1 was the highest expression in the gonad,while Kiss2 was the highest in the brain.4.Injection of Gn RH agonist(LHRH-A)during early sex differentiation can affect the temporal and spatial expression of Gth?,Fshr and Kiss genes,which are key genes of sex axis in Juvenile Sturgeon,(Acipenser schrenckii).Compared with the control group,LHRH-A injection inhibited the expression of Gth? gene in the pituitary of Acipenser schrenckii,while the expression of Gth? gene in the gonad increased first and then decreased.The expression of Gth? in the 3 ?g/kg group reached the maximum value on the 3rd day after injection,significantly higher than that in other groups(P < 0.05).Different from Gth? gene expression pattern,the expression of Fshr m RNA in pituitary and gonad of Juvenile Sturgeon was first increased and then decreased,among which,the expression of Fshr m RNA in gonad of 1 ?g/kg group reached the highest level on the 3rd day after injection,while that of 3 ?g/kg group reached the highest level on the 3rd day after injection.5.The spatial and temporal expression patterns of Kiss1 and kiss2 genes were different in the process of sex differentiation of Acipenser schrenckii.The expression level of Kiss2 was significantly lower than that of Kiss1 at 0-49 days after hatching,and the Kiss1 gene reached its maximum at 21 days after hatching.The expression level of Kiss2 increased from 64 to 199 days after hatching,and decreased after the highest peak at 139 days after hatching,while the expression level of Kiss1 reached the highest peak at 184 days after hatching.In conclusion,the expression of Gth?,Fshr and Kiss are tissue-specific.During the early sex differentiation,LHRH-A can regulate the expression of Gth? and Fshr through HPG axis,so as to participate in the early gonad development and differentiation of Acipenser schrenckii.In addition,Kiss1 and Kiss2 genes are involved in the early sex differentiation and gonad development of sturgeon,and their regulatory functions are different.This study laid a theoretical foundation for further elucidating the molecular regulatory mechanism of sex differentiation and gonad development of Acipenser schrenckii.