Screening And Identification Of Genes Related To Sex Regulation In Asian Yellow Pond Turtle Based On Transcriptome Analysis

Sex determination and regulation mechanisms have always been one of the leading and hot scientific issues in genetics,developmental biology and evolutionary biology,because most vertebrates are dioecious.Vertebrate sex determination mechanisms are diverse.Genetic information and environmental factors are the main factors of sex determination in most vertebrates.According to the causes of sex determination,there are mainly divided into two types,genetic sex determination?GSD?and environmental sex determination?ESD?.Environmental sex determining animals have no sex chromosomes and no genetic differences between males and females.Gender is determined by the environmental conditions of the post-fertilization development process.Many reptiles,some amphibians and fish belong to this way.Environmental factors that affect gender mainly include temperature,humidity,photoperiod,social factors,nutrition,hormones,CO₂,dissolved oxygen,photoperiod,social factors and so on.Temperature-dependent sex determination?TSD?is the most common mode of sex determination in environmental sex determination.The molecular determination mechanism is worthy of further consideration.Vasa,one of the most widely studied germ cell markers,plays a vital role in the development and differentiation of germ cells.The lack of the vasa causes infertility in most male vertebrates,but in flies it causes females infertility.However,little is known about the role of vasa in sex determination of turtles.The Asian yellow pond turtle is an important freshwater turtle species with high edible and medicinal value,and has also been confirmed as a TSD species.High-temperature hatching is biased towards females,and low-temperature hatching is biased towards males.In this paper,the Asian yellow pond turtle was used as an experimental object.First,we compared the transcriptome profiles of females and males and screened a series of differentially expressed genes.Then,we explored the gonad development at different ages and generation process of germ cells of the Asian yellow pond turtles.Last,we conducted a preliminary analysis of the expression pattern of vasa in the generation process of germ cells.The detailed work is as follows:1. Transcriptome analysis of the adult female and male gland of Asian yellow pond turtlesRNA-seq sequencing was performed on the testis and ovaries of adult Asian yellow pond turtles,and a group of genes closely related to sex regulation of Asian yellow pond turtles were screened.such as vasa,dmrt1,cyp17a1,hsf1,sox9,cyp19a1,hsd11b2 and sox8.It lays the foundation for revealing the molecular mechanism of TSD.2. Observation of male and female gonads in different developmental stages.Based on the histological observation of the gonads,we understand the development process and reproductive cycle of the testis and ovary,which provided support for subsequent research on the process of gametogenesis and expression distribution of genes related to sex regulation.3. Cloning and differential expression analysis of vasaThe c DNA sequence of the Asian yellow pond turtle vasa is 2 666 bp in length,including an 11 bp 5'untranslated region?UTR?,a 1 264 bp 3'untranslated region?UTR?,and an open reading frame?ORF?of 1392 bp,encoding 464 amino acids.Fluorescence quantitative results showed that vasa m RNA was specifically expressed in the gonads of adult Asian yellow pond turtles,and the expression level of testis was much higher than ovaries.In embryos,the expression of vasa m RNA can only be detected at the 16th stage,and the level of vasa transcripts in the female temperature?FPT?embryos?33°C?is significantly higher than the male temperature MPT embryos?25??.4. vasa DNA methylation levelsIt was found that the methylation level of the vasa promoter was not significantly different in embryos at different developmental stages of different incubation temperatures,indicating that the increase expression of vasa in FPT embryos may be caused by the proliferation of germ cells.5. Vasa protein expression and distribution in gonads of Asian yellow pond turtlesAccording to the results of fluorescence immunization,Vasa is weakly expressed in spermatogonia,highest in primary spermatocytes,followed by secondary spermatocytes,and then gradually decreased and concentrated in the points of sperm.Sperm cells and somatic cells were hardly detected.In the ovary,Vasa protein is mainly expressed in the early stage of oocytes,but not in the later stages of development.In summary,vasa plays a key role in the differentiation of male germ cells of Asian yellow pond turtles.This study laid the foundation for further discovering the correlation between the expression distribution of vasa and its species-specific functions in interspecific germ cell differentiation.It provides a support for further elucidating the role of vasa in the germ cell differentiation and gametogenesis of turtles,and it is also of great significance for understanding the evolution of the molecular mechanism of TSD.