| Misgurnus anguillicaudatus as a widely distributed small-scale economic fish,occupies a certain proportion in the fish farming industry in China.The existing research shows that decline of wild loach resources has become a crucial factor that restricts the development of the loach aquaculture industry.At present,loach can be divided into three kinds according to the presence and size of its body brindle: big-brindled loach,small-brindled loach,and non-brindled loach,and preliminary breeding studies have shown that they have relatively stable genetic characteristics.Our research group has cloned the full-length cDNA sequence of MaPmch1 gene,MaPomc gene,MaMchr1 gene,MaMc1 r gene and the partial cDNA sequences of the MaMitf gene.Based on previous work,this project has conducted a preliminary study of the molecular regulation mechanism of melanin formation in the loach of Poyang Lake.It is mainly carried out in the following three aspects: The first is the breeding of three kinds of loach,comparing the reproductive performance of them,and the growth index of F1 generation of these three kinds of loach.The second is to detect the expression of five melanin related genes in embryos and larvae at different development periods of loach.Third,the effects of neuropeptide Mch and ?-Msh hormones on melanoma cells were examined and their role in the regulation of melanin synthesis was identified.The main results are as follows:1.In this study,the artificial breeding of the three kinds of loach were conducted.The results showed that the fertilization rate of the group big-brindled female loach × big-brindled male loach was highest,it reached 90.59±4.00%,which was significantly different from that of other groups(P<0.01).This shows that the big-brindled loach has better reproductive performance than the small-brindled and the non-brindled loach.By measuring the growth characteristics of all groups of larvae,it was found that the growth of larvae in small-brindled female loach × big-brindled male loach and small-brindled female loach × non-brindled male loach was significantly faster than that of other groups.At the age of 102 days,the total length and body weight reached 93.12±22.59 mm,5.43±2.98 g and 91.38±27.89 mm,6.70±5.21 g,respectively,which was extremely significant compared with other groups(P<0.01).At the same time,the fitting result of the full-length weight equation is consistent with the results of growth difference analysis.The closer the b-value is to the equidistant growth value 3,the more suitable the pond water environment is for the growth of this group larvae.The growth speed of this group larvae is faster.2.The qRT-PCR technique was used to detect the expression of five melanin related genes in embryos and larvae of 10 different developmental periods of loach.It was found that the expression levels of the MCH,?-MSH,Mchr1,Mc1 r and MaMitf genes began to increase during the early periods of loach embryonic development,such as the Heart-Working period,the MaPmch1,MaPomc,MaMchr1,MaMc1 r and MaMitf gene expression increased to 5.61±0.88 times,2.85±0.89 times,12.36±3.14 times,97.39±28.28 times and 5.67±1.17 times of Multicellular period expression levels.During these periods,the embryos did not have melanin,and all of them were colorless.After the embryos continued to develop into the Pectoral-fin Formation period,the expression levels of the five genes began to increase significantly,respectively reached 16.41±5.18 times,2.91±0.37 times,5.56±0.64 times,44.52±17.79 times and 2.24±1.21 times of Multicellular period expression levels,and the difference was extremely significant(P<0.01).During this period,the loach larvae began to show pigmentation.Therefore,it can be inferred that these five melanin related genes of loach not only participate in the process of melanin synthesis,but also participate in the regulation of embryonic differentiation and energy balance during the early stage of embryonic development.By comparing the ratio of MaPomc/MaPmch and MaMc1r/ MaMchr1,it can be found that the ratio of MaPomc/MaPmch does not change significantly at different developmental stages.However,the ratio of MaMc1r/MaMchr1 changes greatly,which was significantly different from that of multi-cell stage.After the primordial phase of pectoral fins,the ratio reached 7.13 times or more,and there was a significant difference compared with the multi-cell stage.Therefore,it was speculated that during the development of loach embryos and larvae,loach mainly exert its regulation effect by changing the ratio of MaMc1 r gene and MaMchr1 gene expression in vivo.3.The B16-F0 melanoma cell stimulation test results show that stimulation of B16-F0 melanoma cells by neuropeptide Mch can round out the cell morphology,reduce the dendritic cells,and it can inhibit the synthesis of melanin in cells.When the concentration of neuropeptide Mch was 2.5 ?g/mL,the content decreased to 82.59±4.75% of the control group,which was significantly different from the control group(P<0.05).Its receptor gene Mchr1 expression has also been significantly improved.At the concentration of 10 ?g/mL,the expression level increased to 9.55±0.78 times of the control group.Analysis of its Mchr1/Mc1 r ratio found that after the neuropeptide Mch stimulation,melanoma cells Mchr1/Mc1 r ratio increased.At the concentration of 5?g/mL,the ratio increased the most,reaching 3.95±1.54 times of the control group.Contrary to the Mch stimulation test,the ?-Msh melanoma cell stimulation test results show that the number of dendritic melanoma cells increased,and the melanin synthesis of the cells was promoted.At a concentration of 5 ?g/mL,the melanin content reached 140.1±4.8% of the control group.Also,the expression of the receptor gene Mc1 r and the downstream gene Mitf has been significantly improved,At a concentration of 2.5 ?g/mL,it has reached 18.93±5.16 times and 5.12±0.87 times of the control group.And the ratio of Mchr1/Mc1 r decreased significantly after stimulation.After stimulation with the concentration of 2.5 ?g/mL of neuropeptide ?-Msh,the ratio of Mchr1/Mc1 r was significantly decreased,which was 0.24±0.08 times that of the control group,and the difference was extremely significant(P<0.01).In summary,the results of cell stimulation experiments show that the neuropeptides MCH and ?-MSH are involved in the regulation of intracellular melanin synthesis,and that they exert their role in melanin regulation by changing the ratio of intracellular Mchr1 and Mc1 r gene expression. |
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