Study On The Screening,Validation Of Body Color Related Genes And MiRNAs,and The Influence Of Pigments In Chinese Soft-shelled Turtle(Pelodiscus Sinensis)

Body color can not only measure the health of aquatic animals,but also one of the important breeding traits.The colorful skin plays a critical role in the adaptation of aquatic animals to the complex living environment,the mutual recognition within and between species,and species evolution.With the development of bioinformatics technology,a large number of mi RNA have been reported to participate in the body color regulation mechanism of aquatic animals,and the research of mi RNAs is one of the important means to analyze the mechanism of pigmentation and development of aquatic animals.In this study,the Chinese soft-shelled turtle(Pelodiscus sinensis)was used as the research object to analyze the m RNA and mi RNA expression profiles of skin tissues of different colors and skirt tissues at different embryonic development stages,and screened out genes and mi RNAs related to the body color formation and development of the Chinese soft-shelled turtle.The dual luciferase reporter gene was used to verifies the targeting relationship between mi R-18a-3p＿3 and the target gene Sox10.Isolated and cultured Chinese soft-shelled turtle primary melanocytes,and researched the effect of mi R-18a-3p＿3 on the body color related genes of Chinese soft-shelled turtle through cell transfection and in vivo injection.In addition,the carotenoid addition was used to study the effect of exogenous pigments on the body color of the Chinese soft-shelled turtle,hoping to further clarify the body color formation and regulation mechanism of the Chinese softshelled turtle.The main results obtained in this study are as follows:1.Comparative analysis of skin m RNA and mi RNA transcriptome of Chinese softshelled turtleHigh-throughput sequencing analysis was performed on the skin of different colors of the Chinese soft-shelled turtle,1 772 differentially expressed genes and 319 differentially expressed mi RNAs were found in the white skin of the Dongting turtle and the Qingxi black turtle skin.KEGG enrichment analysis found that the differentially expressed genes were significantly enriched in immune-related pathways,suggesting that immune pathways and related genes may be involved in the regulation of adult turtle skin color.Through GO annotation and the differential expression genes of melanogenesis,60 candidate genes related to the skin color of the Chinese soft-shelled turtle were screened.The CDS and 3’UTR regions of the candidate genes were used to predict the targeting relationship with mi RNA,118(including 57 negative correlations and 61 positive correlations)and 122(including 64 negative regulation and 58 positive correlations)m RNA-mi RNA interaction pairs were obtained respectively,of which four interaction pairs have targeted binding sites both in the CDS region and the 3’UTR region.It is speculated that these mi RNAs and genes play an important role in body color formation of Chinese soft-shelled turtle.2.Analysis of m RNA and mi RNA expression profiles in different developmental stages of body color of soft-shelled turtleDuring the embryonic development of the Chinese soft-shelled turtle,the melanocytes begin to develop in the early stage,and their morphology changes from dots to branching and finally to snowflakes.The xanthophore develop in the later stage,and their number is small and their shape is not obvious.Transcriptome sequencing showed that the differentially expressed genes were significantly enriched in linoleic acid metabolism,ECM-receptor interaction,and drug metabolism-cytochrome P450 pathways,72 candidate genes involved in the formation of embryo body color were screened,of which 57 candidate genes formed 1 275 m RNA-mi RNA interaction pairs with 378 mi RNAs,including 737 positive regulatory relationships and 538 negative regulatory relationships.The regulation mode of mi RNAm RNA interaction changes in initial and final development stage.The results of mi RNA family analysis showed that the differential mi RNAs involved in body color formation belong to 64 different mi RNA families,suggesting that these mi RNAs play a significant role in the formation of body color in Chinese soft-shelled turtle embryos.3.The effects of mi R-18a-3p＿3 on genes related to melanin synthesis in Chinese softshelled turtleIn this study,collagenase and trypsin digestion methods were used to isolate and culture soft-shelled turtle melanocytes in vitro.Their morphology is triangular or dendritic.The growth curve results showed that the melanocytes grew accelerated after 6 days,and cell proliferation decreased after 11 days.The body color genes of the melanocytes were expressed.The dual luciferase reporter gene was used to verify the targeting relationship of mi R-18a-3p＿3 and Sox10.The results showed that the activity of luciferase was reduced,indicated that mi R-18a-3p＿3 can target bind Sox10 3’UTR.Cell transfection and vivo injection results showed that mi R-18a-3p＿3 antagomir up-regulated the expression of Sox10,and the expressions of Mitf,Tyr,Tyrp1 and Gpmnb were also up-regulated.Vivo injection also show that the expression patterns of body color genes in different tissues were not the same after mi R-18a-3p＿3 antagomir injection.Among them,the plastron and liver have stronger response effects.The results of the study indicate that mi R-18a-3p＿3 can participate in the regulation of the body color of the Chinese soft-shelled turtle through targeted regulation of Sox10.4.Regulation of nutritional pathways on the body color of Chinese soft-shelled turtleThe juvenile Chinese soft-shelled turtles were fed with different levels of carotenoid addition,and the effects of carotenoids on the growth,antioxidant capacity,body color,carotenoid metabolism genes and antioxidant genes of Chinese soft-shelled turtles were studied.When xanthophyll/canthaxanthin(30/90)mg/kg or 100 mg/kg canthaxanthin added to the feed,the growth performance of turtles can be significantly improved.Diets with high carotenoid levels can increase the serum and liver resistance oxidation capacity.Skin redness,yellowness,and tissue carotenoid content increase linearly with the level of feed canthaxanthin.Supplementation of xanthophyll can increase skin yellowness.The addition of carotenoids can increase the expression of Cat,Sod2 and Bco2.Comprehensive consideration,under the conditions of this study,add 100-120 mg/kg canthaxanthin or 30/90-60/60 mg/kg xanthophyll/canthaxanthin to the feed are best.