| Pelodiscus sinensis(P.sinensis)is a famous and excellent species for aquaculture in China,and its belongs to the genus Reptilian,Turtle and Brassica.P.sinensis has a Z/W sex chromosome and is a genetic sex determination(GSD).In the breeding process,male individuals have obvious traits and economic advantages(fast growth,large individuals,wide skirts,rich nutritional value,high market unit price),Therefore,gender control and breeding of full males will be an important development direction of the P.sinensis breeding industry.The key to achieving this goal is to analyze the molecular mechanisms of gender determination and differentiation.The R-spondin1(RSPO1)gene is a member of the R-spondins family and encodes a protein containing a type 1 thrombospondin repeat(TSR-1).And it is considered to be a regulator of vertebrate ovarian development.Related studies have shown that RSPO1 gene which is considered to be an important regulator of mammalian and fish ovarian development,and its mutation causes female to male reversal,but the role in the sex determination of reptiles is unclear.In this study,we cloned cDNA full-length of the P.sinensis RSPO1 gene,and its expression pattern was analyzed.The function deletion was identified by RNA interference in vivo.The results are as follows:(1)RSPO1 full sequence cloning and evolution analysisThrough RACE technology,we cloned the full length of the RSPO1 gene,full length 1975 bp,its including 33 bp 5' non-coding region,738 bp open reading frame,1204 bp 3' non-coding region,encoding 245 amino acids.The homology analysis showed that the amino acid sequence of the RSPO1 gene of P.sinensis was 94% similar to that of the Trachemys scripta.The phylogenetic tree is divided into two subgroups,one for fish and the other for birds,amphibians,reptiles and mammals.Among them,the P.sinensis has the closest relationship with the Trachemys scripta,and has thefurthest relationship with the Oryzias latipes.(2)Expression pattern of RSPO1 and expression changes in sexual reversalqRT-PCR showed that the expression level of RSPO1 in adult ovary was the highest,which was significantly higher than that in testis,heart,liver,spleen,lung,kidney,muscle and small intestine.In the 14 th stage of embryonic development of P.sinensis(before gonadal differentiation),RSPO1 gene has been expressed in gonadal tissues,and the female expression level is significantly higher than that of males.In the subsequent sex determination period(15-20),RSPO1 maintained this trend of high female expression.During the reversal of females induced by aromatase inhibitors(15th and 16 th stages),the expression level of RSPO1 in ZW gonads was significantly down-regulated at the 17 th stage,which was close to the expression level of ZZ gonads in the control group.This indicates that the RSPO1 gene is involved in the initiation of female sex differentiation in P.sinensis.(3)Loss of function of RSPO1We performed a functional deletion verification of the RSPO1 gene by RNA interference.Histological observations showed that the morphology and gonadal structure of some ZW embryos showed obvious male characteristics after RSPO1 knockdown.Although the cortical area did not degenerate to a male-like monolayer,the medulla was obviously developed,and male sex was observed.At this time,the gonads present an intersexual feature dominated by male traits.At the same time,qRT-PCR showed that the expression of Foxl2,a key gene of ovarian development,was significantly down-regulated in the ZW gonads after RSPO1 knockdown,while the expression of Dmrt1 was up-regulated in males.Immunofluorescence staining further showed that after RSPO1 knockdown,a large number of male SOX9 protein signals were detected in the sex cord cells of the ZW medulla,and the distribution of germ cells was observed in the medullary region.Although a small number of germ cells were still present in the cortical area,the number was significantly less than the number of germ cells in the ZW gonad cortex of the control group.The above data prove that knocking down RSPO1 can cause female to male reversal to some extent before the differentiation of P.sinensis gonads.We cloned the full-length cDNA of the RSPO1 gene of the P.sinensis was cloned and the expression pattern of the embryonic gonadal development(high femaleexpression)was revealed.It was found that RSPO1 deletion can induce partial reversal,This indicates that RSPO1 participates in the sex determination and differentiation process of P.sinensis and is an important regulator of ovarian development.This study laid the foundation for the molecular mechanism research of sex determination and differentiation of P.sinensis,and provided a theoretical basis for the development of male control technology. |
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