| Sea cucumber(Apostichopus japonicus)belong to the Echinopermata,Eicutherozoz,Holothuroidea,Aspidochirotidae,Sitchopodidae.In the past five years,the aquaculture production of sea cucumber in China accounted for about 1% of the national mariculture economy,and its output value accounted for 12.9% of the total output value of mariculture in China.Significant gender differences were found in individual growth rate,immunity and additional economic value of sea cucumber.Sea urchin(Mesocentrotus nudus)belong to the Echinopermata,Echinoidea,Camarodonta,Strongylocentrotidae.Sea urchin is an important mariculture species in China,and its gonads are the only edible and the most efficient value part.Male sea urchin has obvious advantages in growth speed,immune ability,gonadal nutrients,etc.At present,the gonadal development mechanism of echinoderm including sea cucumber and sea urchin is always a mystery.Mining gonadal development-related genes is part of the effective ways to analyze the gonadal development mechanism.Studies have shown that the Dmrt1 and Sox E genes play important regulatory roles in sex determination,gonadal differentiation,and maintenance in male vertebrates.In invertebrates,there is little research on the gene function and interaction between Dmrt1 and Sox E.In this study,the Sox E gene sequences of sea cucumber and sea urchin were firstly identified in the genome and gonadal transcription by homologous alignment,and then the full length of its c DNA was cloned using the rapid amplification of c DNA ends(RACE)and the molecular characteristics and evolutionary relationship were analyzed.Subsequently,the dynamic expression changes in different adult tissues,altered gonadal development stages and embryonic development stages were elucidated by real-time quantitative PCR(RT-q PCR).Finally,RNA interference(RNAi)was utilized to analyze the function of Sox E gene in testis development of sea cucumber and the interaction relationship between Dmrt1 gene and Sox E gene was explored.The main research results are as following:(1)The full-length c DNA of the Sox E gene(Mn Sox E)of sea urchin was 2687 bp,with5’-UTR 470 bp and 3’-UTR 726 bp,and an Open Reading Frame(ORF)1491 bp,totally encoding 496 amino acids.Domain prediction results showed that there was a conservative HMG domain between amino acid residues 96-166 in the protein sequence of Mn Sox E.(2)The full-length c DNA of the Sox E gene(Aj Sox E)of sea cucumber was 3389 bp,with5’-UTR 275 bp and 3’-UTR 1734 bp,and an Open Reading Frame(ORF)1491 bp,totally encoding 496 amino acids.Domain prediction results showed that there was likewise a conservative HMG domain between amino acid residues 85-155 in the protein sequence of Aj Sox E.(3)The Mn Sox E was highly expressed in the gonad tissues,and its expression in the testis was 5.05 fold that in the ovary,and it was slightly expressed in the tube feet and intestines.In addition,Mn Sox E was not expressed in the early embryonic development stage and its transcript was initially detected when it developed to 4-carpal.(4)The Aj Sox E was most expressed in the tubules,and a large number of transcripts could be identified in the ovary,longitudinal muscle and respiratory tree.Different from vertebrates,the expression level of Aj Sox E in the ovary was significantly higher than that in the testis,and the expression level in the ovary was 34.75 f old that in the testis,and the expression levels were significantly different at different stages of gonadal development.In addition,a large number of transcripts of the Aj Sox E was detected when the embryos developed to blastula.(5)Using RNAi to knock down the Aj Sox E in male,it was found after the Sox E was knocked down,the expression of Dmrt1 gene dramatically decreased to an almost undetectable level,and the expression of germ cell marker gene piwi was also significantly decreased,but the expression of foxl2 gene located in the female sex differentiation pathway increased significantly.Knocking down the Aj Dmrt1 caused the down-regulation of Sox E expression in the testis(45.52%–88.65%),down-regulation of piwi expression(0.97%–58.36%)and up-regulation of foxl2 gene expression(66.75%–117.41%).In summary,this study clarified the molecular characteristics and expression characteristics of Sox E in Mesocentrotus nudus,revealing that its molecular characteristics were highly conservative in evolution,and were differentially expressed in the testis and ovary tissues,suggesting that Mn Sox played an important role not only in the testis development of Mesocentrotus nudus,but also in the ovary development.Similar to the Mn Sox E,the molecular structure of Aj Sox E was evolutionarily conservative.Surprisingly,the expression level of Aj Sox E in ovary was significantly higher than that in testis,suggesting that Sox E might play an important regulatory role in the development of ovary and testis in Apostichopus japonicus.The knock-down of Aj Sox E in male caused the down-regulation of Dmrt1 gene and piwi gene expression,while the up-regulation of foxl2 expression,suggesting that Aj Sox E was essential for maintaining the testis differentiation state.Interestingly,knocking down the Aj Dmrt1 gene in male also caused the down-regulation of Sox E gene and piwi gene expression,while up-regulation of foxl2 expression,suggesting that the interaction between Dmrt1 gene and Sox E gene played an important regulatory role in maintaining the differentiation and development of the testis in Apostichopus japonicus.This study laid a foundation for analyzing the sex determination and differentiation mechanism of echinoderm. |
