Transcriptome And Expression Analysis Of Foxl2 And Cyp11b In Embryonic Gonads Of Mauremys Mutica

The mechanisms of sex determination and regulation are one of the most frequently asked scientific questions in genetics,developmental biology,and evolutionary biology.In vertebrates,the mechanism of sex generation is influenced by a variety of factors.Genetic information and environmental factors dominate and are usually divided into two categories:genetic sex determination（GSD）and environmental sex determination（ESD）.There is no genetic difference between the sexes of ESD animals,and their sex depends on the environmental conditions during their embryonic development,such as temperature,humidity,nutrition,hormones,CO₂,dissolved oxygen,photoperiod and other environmental factors.Among them,temperature-dependent sex determination（TSD）is the most common mode of sex determination in ESD.The Asian yellow pond turtle（Mauremys mutica）is a national second-class protected wild animal,and has high application value in ornamental,edible,medicinal and scientific research.The sex of Mauremys mutica is regulated by temperature,and the individual differences between males and females are large,showing the phenomenon that the female is larger and the male is smaller.At the same time,Mauremys mutica belongs to the intermediate species transitioning from invertebrates to vertebrates,and it is an ideal model for studying temperature-dependent sex determination in vertebrates.So far,there are few reports on the gonad development and sex differentiation of Mauremys mutica,especially the molecular mechanism of sex regulation is still lacking.In this study,Mauremys mutica was used as the experimental object,and the transcriptomes of six embryonic developmental stages of male-producing temperature（MPT）and female-producing temperature（FPT）were compared and analyzed.A series of potential candidate genes in response to temperature changes were screened.Because Mauremys mutica is a TSD animal,it has sex dimorphism,and there are large differences between males and females,the star genes（Foxl2 and Cyp11b）that have been reported for sex determination or sex differentiation of TSD animals were selected as entry points.The two genes,Foxl2 and Cyp11b,were cloned and identified,expression analysis and cell localization research were carried out,so as to preliminarily analyze the biological function of Foxl2 and Cyp11b in the sex determination or sex differentiation of Mauremys mutica.The detailed research results are as follows:1.Comparative transcriptome analysis of embryonic gonads of Mauremys muticaWe first performed transcriptomic analysis in six embryonic developmental gonads of Mauremys mutica MPT and FPT,and screened out several classes of candidate genes that respond to temperature changes,such as heat shock protein family genes（Dnajb6a,Dnaja4,Hspa8 and Hsp90aa1）,temperature sensor genes（Mmp17,Mmp28,Tmco6,Gria3 and Eif3f）and genes related to sex differentiation and gonadal development（Tex15,Insr,Igf1r,Cirbp,Esr1,Dmrt2 and Serpinh1）.At the same time,the gene expression similarity and time-course difference in the gonad tissue of MPT and FPT in different stages of MPT and FPT of Mauremys mutica and other two reported TSD turtles（Trachemys scripta elegans and Chrysemys picta bellii）were compared and analyzed.Eight shared genes were found in these three TSD turtles,suggesting that the three may share a common gonad-specific regulatory mechanism.This will provide basic data for the later elucidation of the sex determination and sex differentiation mechanism of Mauremys mutica and other TSD turtles.2.Cloning and sequence analysis of Foxl2 and Cyp11b in Mauremys muticaThe c DNA sequences of Foxl2 and Cyp11b were cloned by RACE technology.Among them,the sequence length of Foxl2 c DNA is 2,574 bp,including 621 bp 5’non-coding region（UTR）,1,047 bp 3’non-coding region（UTR）and 906 bp open reading frame（ORF）,encoding a total of 301 Amino acid;the sequence length of Cyp11b c DNA is1,305 bp,including 13 bp 5’non-coding region（UTR）,15 bp 3’non-coding region（UTR）and 1,277 bp open reading frame（ORF）,encoding a total of 434 amino acid.Multiple comparison analysis and phylogenetic tree of amino acid sequences were carried out.The results showed that the Foxl2 of Mauremys mutica had the highest homology with Mauremys reevesii.The fishes have a far-reaching relationship;the Cyp11b of Mauremys mutica has high homology with Chrysemys picta bellii Trachemys scripta elegans,and the similarity is as high as 99.54%and 98.62%.The kinship of Trachemys scripta elegans and Mauremys reevesii is relatively close,and the kinship with humans and mice is farther away.The protein tertiary structure prediction results of Foxl2 and Cyp11b showed that they each had one conserved domain.3.Expression and cellular localization analysis of Foxl2 and Cyp11b during embryonic development of Mauremys muticaThe RT-q PCR results showed that among the adult tissues of Mauremys mutica,the expression level of Foxl2 was the highest in the ovarian tissue,on the contrary,the expression level of Cyp11b was the highest in the testis tissue,and the expression level of the two were very similar in other tissues.In the gonads of FPT and MPT embryos,the expression of Foxl2 in FPT embryo gonads was significantly higher than that in MPT embryo gonads,while Cyp11b was just the opposite,its expression in MPT embryo gonads was significantly higher than that in FPT embryo gonads.Sectional observation of embryonic gonads of MPT and FPT at different developmental stages showed that the medulla region of the embryonic gonads of MPT was more developed,with a typical primitive sex cord structure（containing a large number of germ cells）,while its cortical region very thin（only 1 layer of cells）;in contrast,the cortical area of the embryonic gonads of FPT is more developed（containing a large number of germ cells）and thicker（about 3-4 layers of cells）,while the medulla area the degeneration is severe,with partial hollowing and few or no germ cells.The results of in situ hybridization at different embryonic stages of MPT and FPT showed that the chromogenic signal of Foxl2 m RNA was mainly expressed in the cortex of the gonads of FPT embryos,while the medulla had little or no signal;In contrast,the chromogenic signal of Cyp11b m RNA was mainly expressed in the medulla region of the MPT embryonic gonad,while the cortical region showed little or no signal;The antisense probes of Foxl2 and Cyp11b both showed blue-purple positive signals,and the sense probes of the control group were colorless and colorless.To sum up,Foxl2 and Cyp11b are involved in the process of sex differentiation and gonadal development of Mauremys mutica.This lays the foundation for further elucidation of other molecular mechanisms of temperature dependence of Mauremys mutica.