Bioinformatics Analysis Of WNT4,β-catenin And DAX1 Gene And The Correlation Of Gonadal Development In Haliotis Discus Hannai.

Haliotis discus hannai is known as the "crown of Marine treasures" among seafood because of its large size,high yield and rich nutrition.In recent years,the yield of abalone in China has been increasing year by year,but it was found that the gonadal development of abalone was not balanced in the process of breeding,which had adverse effects on the subsequent breeding and breeding.The main research contents of this paper are as follows:After gene sequences were obtained from the transcriptome library and homologous sequences constructed in the early stage,bioinformatics analysis was carried out,and then the expression patterns of WNT4,β-catenin and DAX1 genes in different tissues of male and female Abriotis platyphalis in different months were analyzed by fluorescence quantitative method.Finally,the expression of WNT4,β-catenin and DAX1 genes in the gonads of male and female Abalone discosus in different months was analyzed by in situ hybridization.The results of this study provide basic data for further understanding the developmental mechanism of gonadal glands and exploring the functions and regulatory mechanisms of gonadal development related genes in Abalone discosus.The main research results are as follows: The main research results are as follows:1 Bioinformatics analysis of WNT4 gene and the correlation of gonadal development in H.discus hannai.As an important member of the WNT gene family,WNT4 plays a pivotal role in development processes of many animals.In the present study,the c DNA sequence of WNT4 gene was obtained from the transcriptome library of Haliotis discus hannai.The length of the WNT4 gene was 2002 bp,including5’-UTR 342 bp and 3’-UTR 595 bp.The open reading frame(ORF)was 1071 bp,encoding a polypeptide of 356 amino acids.As revealed in the real-time quantitative PCR analysis in different tissues of male and female abalone in different months,WNT4 gene was expressed in gonad,muscle,mantle and gill,but hepatopancreas.The expression level of WNT4 gene in gonad of female was the highest in July(P<0.05),followed by May and August,and it was significantly higher than that in June and September.There were no significant differences between May and August,and June and September.In gonads of male individuals,the expression of WNT4 in different months was in a descending order of July > August > September > May > June.The expression level in July and August were significantly higher than that in other months,and that in September was significantly higher than that in June,but not significantly different from that in May.In addition,the expression level of WNT4 gene in testis was higher than that in ovaries.In situ hybridization results showed that WNT4 had obvious blue-purple hybridization signals in male and female gonads in different months.The expression characteristics of WNT4 in many tissues of H.discus hannai indicate that it has a wide range of tissue expression characteristics and participates in the life process of many tissue cells in the form of signal molecules.From the gonad expression characteristics,it is speculated that it may play a role in the development of gonadal development.2 Bioinformatics analysis of β-catenin gene and the correlation of gonadal development in H.discus hannai.β-catenin,a member of the catenin family,is an important transcriptional effector in the classical WNT signaling pathway.In this study,the core sequence was amplified by homologous sequence alignment,and then the full-length c DNA sequence of β-catenin gene was obtained by RACE technique.The length of the c DNA sequence was 3083 bp,including a 5 ’UTR of 100 bp and a 3’ UTR of 1219 bp,among which the ORF length was1761 bp,encoding 587 amino acids.The sequence was homologous with P.canaliculata,C.fornicata,A.californic,P.fucata and C.gigas,respectively is 90.47%、89.70%、87.91%、86.97% and 85.50%.QRT-PCR was used to analyze,the results showed thatβ-catenin gene was expressed in mantle,muscle,gonad and gill in different months,except hepatopancreas.In female gonads,the expression of β-catenin gene was the highest in July.In male gonads,the expression level of β-catenin gene in different months was May < June < August < September < July.In addition,the expression of β-catenin gene in ovary was higher than that in testis.The results of ISH showed that there were obvious blue-purple hybridization signals in the female gonads of different months,mainly located in the cytoplasm,nucleus and somatic cells of oocytes.There were also blue-purple hybrid signals in male gonads in May,June,July and August,mainly located in connective tissue and spermatocyte,but no blue-purple signal was detected in September gonads.The hybridization signal was strongest in both male and female gonads in July.There was no signal in the control group.3 Bioinformatics analysis of DAX1 gene and the correlation of gonadal development in H.discus hannai.DAX1 protein is a kind of lonely,belongs to the nuclear receptor NR,an important member of the super family,this study obtained from wrinkles dish bao transcriptome library DAX1 gene c DNA sequence,the sequence is 2835 bp long,including 5 ’the translation section(5’ UTR)54 bp,the 3’ UTR of 1705 bp,the ORF length of 1077 bp,coded 358 amino acids.The protein sequence was associated with A.californic,A.farreri,C.virginica,C.gigas,M.yessoensis and P.canaliculata the homology of canaliculata was 64.63%,62.11%,59.77%,59.38%,53.45% and 50.33%,respectively.QRT-PCR was used to analyze,the results showed that,except hepatopancreas,DAX1 gene was expressed in mantle,muscle,gonad and gill of male and female abalone discus in different months.In female gonads,the expression of DAX1 gene was the highest in July.There were significant differences between May,June,August and September.In male gonads,the expression level of DAX1 gene from low to high was September,August,June,May and July.The results of ISH showed that there were obvious blue-purple hybridization signals in the female gonads of different months,mainly located in the cytoplasm,nucleus and oocytes of oocytes.There were also blue-purple hybrid signals in male gonads in May,June and July,mainly located in spermatocyte and connective tissue,but the signal was weak,and no blue-purple signal was detected in male gonads in August and September.The hybridization signal of female gonads was stronger than that of male gonads.