| Chinese perch(Siniperca chuatsi) is one of the most valuable freshwater fishes in China and has vital economic value in China.At present,there are few reports on the regulation of neuropeptide on the reproductive physiology of Chinese perch.In this study,extracting total RNA from the brain tissue of Chinese perch,the complete cDNA sequence of KISS-1 and NUCB2 genes were cloned by using PCR amplification and RACE techniques,with immunofluorescence double staining technology and real-time fluorescence quantitative PCR technology,it analyzes Kisspeptin controlled by the above two genes and the distribution of Nesfatin-1 in the early embryonic development of Chinese perch,as well as the expression of m RNA in different gonadal development stages.The aim of this study is to explore the effects of the neuropeptides Kisspeptin and Nesfatin-1 on the growth and development of Chinese perch from the perspective of neuroendocrine,and provide basic theoretical data for perfecting and improving the artificial breeding and breeding technology for Chinese perch.(1)The complete cDNA sequence of KISS-1 gene was obtained by PCR amplification and RACE technique,with a total of 318 bp,encoding 105 amino acid residues,a signal peptide of the first 20 amino acids,a total protein molecular weight of 11675.46,and a theoretical isoelectric point of 9.93.The core functional area of Kisspeptin in the Chinese perch,is predicted to have a sequence of Kisspeptin-10 of YNLNSFGLRY-NH2,which is highly conserved among vertebrates.(2)By PCR amplification and RACE cloning,the complete cDNA sequence of NUCB2 gene with a length of 1755 bp is obtained,which contains 5 ′ untranslated region of 80 bp,1467bp coding region,and 3 ′ untranslated region of 209 bp.A total of488 amino acid residues are encoded,the first 23 amino acids were the signal peptide region.The total protein molecular weight is 57639.79 and the theoretical isoelectric point is 4.86.The peptide region can be divided into 82 amino acid Nesfatin-1,71 amino acid Nesfatin-2 and 308 amino acid Nesfatin-3.The M30 region of Nesfatin-1,has the same amino acid sequence in fish,which is highly conserved in Vertebrata and fish.(3)As the results of immunofluorescence double staining shows,Kisspeptin and Nesfatin-1 began to express in the blastocyst stage of Chinese perch,and in the premembrane stage,the two neuropeptides migrated to different germ layers of the placenta with the development of the embryo.It was distributed in eyes,organs,caudal stalk and pectoral fin.The qPCR results also saw that the m RNA expression of Kisspeptin and Nesfatin-1 changed during the different stages of embryonic development.The expression level of Kisspeptin was significantly lower than that of the opening feeding period in all periods before the yolk sac was not consumed;The whole stage is low,and it is significantly lower in the opening period than other embryonic development periods.Immunofluorescence showed that the difference in the distribution of Kisspeptin and Nesfatin-1 before membrane exit and the almost uniform distribution in the opening feeding period and qPCR detected that the expression of Kisspeptin m RNA in the opening period was significantly up-regulated(P <0.05),and Nesfatin-1 was significantly down-regulated(P <0.05),proving that there are temporal and spatial differences between the two genes in the regulation of Chinese perch embryo development.(4)The expression of Kisspeptin and Nesfatin-1 m RNA in tissues of stage Ⅱ of male and female Chinese perch was detected by qPCR technology.The results showed that Kisspeptin had the highest expression in female liver and gills(P <0.05),and the highest expression in male testis(P <0.05)),but with low expression in the brains of female and male fish(P <0.05),and there are gender dimorphisms in liver,gonads,intestines,gills and middle kidney tissues.Nesfatin-1 has the highest expression in the liver of female and male Chinese perch,and it is also highly expressed in the testis of male fish.There is gender dimorphism in the expression of gonadal tissues,and the difference in the expression of other tissues and organs is not obvious.It is initially confirmed that Kisspeptin-1 and Nesfatin-1 play a regulatory role in Chinese perch energy regulation and gonad development.(5)To detect the expression of Kisspeptin and Nesfatin-1 in the brain,liver,and gonads of male and female Chinese perch gonad development stage Ⅲ-Ⅴ by using qPCR technology.The results showed that Kisspeptin was significantly overexpressed in the gonads(P <0.05),and was significantly upregulated in the ovary as the gonadal developed(P <0.05),and the testis was significantly downregulated(P <0.05).The expression was significantly up-regulated(P <0.05),and also significantly increased in the liver and brain of Ⅳ males(P <0.05).Nesfatin-1 had the highest expression in the liver of female and male Chinese perch in stages Ⅲ-Ⅴ(P <0.05),and also had higher expression in the testis.The relative expression of Nesfatin-1 in male Chinese perch was low,and there was no significant change in temporal expression(P> 0.05).It is further confirmed that Kisspeptin and Nesfatin-1 may be directly involved in regulating the energy metabolism and gonadal development of Chinese perch. |
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