Cloning And Expression Of Gnih/Gnihr3 Genes In Different Ploidy Cyprinids

The allotetraploid hybrids(4n=200)were obtained by mating red crucian carp(Carassius auratus red var.)(2n = 100,♀)and the common carp(Cyprinus carpio L.)(2n=100,♂).Using improved allotetraploid hybrids as the male parent and diploid red crucian carp as the female parent,sterile allotriploid crucian carp(Xiangyun crucian carp II)was obtained through chromosome ploidy manipulation technique.Sterile triploid fish provides good experimental material for the study of the gonadal development regulation.Gn IH(gonadotropin inhibitory hormone)has been proven to be a hypothalamic neuropeptide that regulates the reproductive axis negatively in birds and mammals,it binds to the receptor(gonadotropin inhibitory hormone receptor,Gn IHR)and effects together to inhibit the synthesis of Gt H(gonadotropin).Currently,there are few studies on the expression and mechanism of Gn IH/Gn IHR system in fish.The full-length complementary DNAs(c DNAs)of the gnih and gnihr3 gene in diploid red crucian carp and Xiangyun carp Ⅱ were obtained.And then the comparative analysis on the expression of gnih and gnihr3 was carried out in red crucian carp and Xiangyun crucian carp II by Real-time quantitative PCR(q PCR)and tissue in situ hybridization,which provided the theoretical basis on the research of fish reproductive physiology and breeding.We have obtained the main results as follow:1.The sequence of gnih/gnihr3 genes in red crucian carp and Xiangyun crucian carp II were cloned and analyzed.The full-length c DNAs of gnih gene in red crucian carp and Xiangyun crucian carp II were 765 bp in size with 5’ UTR(untranslated region)of 27 bp,3’ UTR of 144 bp and ORF(open reading frame)of 594 bp that encoded 197 amino acids.The full-length c DNA of gnihr3 gene in red crucian carp was 1678 bp with 5’ UTR of 130 bp,3’ UTR of 186 bp and ORF of 1362 bp that encoded 453 amino acids.The full-length c DNA of gnihr3 gene in Xiangyun crucian carp II was 1670 bp with 5’ UTR of 130 bp,3’ UTR of 186 bp and ORF of 1362 bp that encoded 453 amino acids.The amino acid sequences of Gn IH of red crucian carp and Xiangyun crucian carp II both contained the typical LPXRF(X = L or Q)marker of Gn IH peptide.Gn IHR3 was a G-protein coupled receptor with seven transmembrane domains.Multiple alignments of amino acid sequences and phylogenetic tree analysis showed that Gn IH/Gn IHR3 protein of red crucian carp and Xiangyun crucian carp II had high homology with other fish Gn IH/Gn IHR3 protein and they had a closer evolutionary relationship,but the evolutionary distance from birds and mammals Gn IH/Gn IHR3 protein was long.2.The expression of gnih/gnihr3 genes in the tissues of red crucian carp and Xiangyun crucian carp II was studied.QPCR technology was used to detect the expression of gnih/gnihr3 genes in 32 tissue samples of two kinds of fish(♀,♂).The results showed that:(1)The gnih gene was expressed the highest in the hypothalamus of these two kinds of fish,there was no significant difference in expression in the hypothalamus of male and female red crucian carp.However,the expression level of gnih gene in the hypothalamus of female Xiangyun crucian carp II was significantly higher than that of male.(2)The gnihr3 gene was clearly expressed on the hypothalamic–pituitary–gonad axis(HPG)of male and female red crucian carp,especially in testes,the expression level of gnihr3 was very high.As for Xiangyun crucian carp II,the gnihr3 gene was highly expressed in the gonads of the male and female.It showed that gnih is mainly expressed in the hypothalamus,combining with the receptors distributed in HPG widely and acting through multiple pathways.3.The expression of gnih/gnihr3 genes in the reproductive axis of red crucian carp and Xiangyun crucian carp II was studied.A comparative study on the expression of gnih/gnihr3 genes in female diploid red crucian carp and triploid Xiangyun crucian carp II reproductive axis was carried out using q PCR technology.Research results showed that:(1)The expression level of gnih gene in the hypothalamus of red crucian carp was higher than that of Xiangyun crucian carp II.However,the expression levels of gnih were lower in the ovaries and pituitary of these two kinds of fish,and there was no significant discrepancy.(2)The expression level of gnihr3 gene in the hypothalamus and pituitary of red crucian carp was higher than that of Xiangyun crucian carp II,opposite,the expression level of gnihr3 gene in the ovary of red crucian carp was lower than that of Xiangyun crucian carp II.Studies have shown that the expression level of gnrh2 in the brain of Xiangyun crucian carp during the non-breeding season was higher than that of red crucian carp,it may be related to the low expression of gnih and its receptor in allotriploid crucian carp.4.Localization of gnih/gnihr3 genes in brains and pituitaries of red crucian carp and Xiangyun crucian carp II was studied.Strong gnih hybridization signal can be observed in Dc,Dld,Dd,Npp,Npo,NLT in the brain of diploid red crucian carp and triploid Xiangyun crucian carp II by tissue in situ hybridization.gnih hybridization signal in the brain of triploid Xiangyun crucian carp II was weaker than that of red crucian carp.Strong gnihr3 hybridization signals were observed mainly in the PPD and RPD regions of the pituitary,and weak hybridization signals in NH and PI regions.Besides,the signal of gnihr3 hybridization of triploid Xiangyun crucian carp II in the pituitary was much weaker than that of diploid red crucian carp.