Cloning And Identification Of Nile Tilapia Jarid2 Gene And The Effect Of High Temperature Induction On Its Expression Change

There are three types of gender determination in lower vertebrates: genotype sex determination(GSD),environmental sex determination(ESD),and genetic-environmental sex determination(GSD + ESD).Among them,the effect of temperature on environmental sex determination is more critical.In temperature-dependent sex determination,reptiles have been studied in depth,but studies on fish have been scarce.Some studies have found that temperature is the most important and critical environmental factor in sex determination in fish.Many fish belong to the genetic-environmental sex determination(GSD + TSD)type.The sex determination of Nile tilapia belongs to the gene-temperature dependent sex determination method.It is not only controlled by genes but also affected by temperature.Nile tilapia is the largest farmed fish exported in China.The growth rate and reproductive capacity of males are significantly higher than that of females.Increasing the percentage of males in the breeding process can effectively increase production.Therefore,studying the Nile tilapia sex reversal is of great significance to improve its economic benefits.Jarid2 is a sex-related gene,and Jarid2 has been shown to be related to temperature sex determination in studies on turtles,but there have been no related studies in Nile tilapia.In the process of sexual reversal from female to male,how has its sex-related gene Jaird 2 changed? The study of this problem will help to reveal the mechanism of high temperature-induced sexual reversion,provide a theoretical basis for increasing the male rate of Nile tilapia.Based on this,this study intends to perform high-temperature treatment at the critical period of gonad differentiation of Nile tilapia to produce physiological males,and to study the temporal and spatial variation of Jarid2 in this process.This study grouped Nile tilapia 21 days after fertilization(9dpf + 12d): control female group(XX,normal breeding at 28 ° C),control male group(XY,normal breeding at 28 ° C),and high-temperature group(XX + HT,36 ° C high temperature treatment).The differences in the expression levels and positions of Jarid2 in each group were analyzed to reveal the changes of the sex-related gene Jarid2 during sexual reversal.The results are as follows:1.In this study,the complete sequence was obtained by sequencing the Jarid2 gene after ligation and transformation,and the sequence was compared with the NCBI sequence.The alignment found that the sequences could be correctly matched.2.In this study,the quantitative analysis of Jarid2 in Nile tilapia was carried out by quantitative Real-time PCR(qRT-PCR).It was found that the content in blood was the highest,and the difference in other tissues was not significant.The content of Jarid2 in the brain of male fish is higher than that in female brain,and the expression of Jarid2 in testis is significantly higher than that in ovaries.3.In this study,Western Blot was used to detect the protein level of 11 tissues in Nile tilapia.The study found that the Jarid2 protein content in blood was the highest,and the Jarid2 content in male brains was also higher than that in female brains.The expression of Jarid2 in testis was significantly higher than that in ovaries.These are consistent with the results of quantitative Real-time PCR(qRT-PCR).4.The expression of Jarid2 mRNA in the gonads of Nile tilapia control female group(XX,28 ° C normal breeding),control male group(XY,28 ° C normal breeding)and high temperature treatment group(XX + HT,36 ° C high temperature treatment)was detected by quantitative Real-time PCR.The expression level of Jarid2 mRNA in the gonads.The expression level of Jarid2 mRNA in the XY group was the highest,followed by the XX + HT group,and the expression of Jarid2 mRNA in the XX group was the lowest.5.In this study,the Western Blot method was used to test the control female group,the control male group and the high temperature treatment group.The results showed that the male fish Jarid2 protein expression was the highest,followed by the high temperature treatment group,which were significantly higher than the control female fish group,which is basically consistent with the results of quantitative Real-time PCR.6.In this study,the immunohistochemical technique was used to detect the differences in expression levels and locations of the gonads Jarid2 at 21 days after fertilization in the control female group,the control male group,and the hyperthermia group.The results showed that the expression levels of the control male group and the high temperature treatment group were similar and significantly higher than those of the control female group.In summary,the female Nile tilapia treated with high temperature experienced a sexual reversion from female to male.During this process,the expression of the sex-related gene Jarid2 also changed to some extent,the expression level from the control female was similar to that of the control male.However,the transformation of cell types in its gonads requires further research.