Expression Difference Of CYP19A1 Between-sex And-ploidy In Loach Misgurnus Anguillicaudatus And Its Relationship With DNA Methylation

Cytochrome P450 aromatase（P450arom）,one of the steroidogenic enzymes,encoded by cyp19a1 gene is the key enzyme of the aromatase complex that catalyzes the conversion of androgens into estrogens.It is found that cyp19a1 gene not only plays a key role in sex determination of fish,but also regulates the growth and development of fish.The loach Misgurnus anguillicaudatus（Cobitidae,Cypriniformes）is a common small economic fish in China.There are not only diploids in wild loach,but also a large number of tetraploids in the Yangtze River Basin.The tetraploid loach has significant growth advantage compared with diploid loach,but its gonad development is relatively backward.Previous studies in our laboratory found that in the same environment,the diploid loach can reach sexual maturity at1 year old and begin reproductive activities,while the tetraploid loach can reach sexual maturity after 2 years old.In addition,both diploid and tetraploid,the growth and body size of female loach were better than that of male,which showed obvious gender dimorphism.In order to study the correlation between the expression of cyp19a1 gene and these biological characteristics,the full-length c DNA and 5’-flanking sequence of cyp19a1 gene in loach were cloned and bioinformatics analysis was carried out.The expression differences of cyp19a1between sex at different developmental stages and its correlation with dnmts expression level were compared in diploid loach.The changes of blood estradiol and testosterone levels in the corresponding developmental stages were analyzed.And the correlation between the expression level of cyp19a1 gene and DNA methylation level was studied.Finally,the knockout strains F₀of cyp19a1a/cyp19a1b were constructed to prepare experimental fish materials for further study.The main results were showed as follows:1.Cloning and analysis of full-length c DNA and 5’-flanking sequence of cyp19a11)The length of the c DNA sequence of cyp19a1a in loach was 1905 bp,which contained29 bp 5’UTR,301 bp 3’UTR and 1575 bp ORF,encoding 524 amino acids.Some important functional domains were observed in the amino acid sequence,such as I-helix region,aromatase specific conservative region and heme binding region.A 2040 bp 5’-flanking sequence was obtained by hi TAIL-PCR method,which contained the typical element TATA-box,and some important transcription factor binding sites such as SP1,AP-1,C/EBPβ,SRY,ER,CREB,GR,and et al.2)The loach cyp19a1b was 1666 bp in length,including a 39 bp 5′UTR,a 124 bp 3′UTR,and a 1503 bp open reading frame,encoding 500 amino acids.Consistent with other species,cyp19a1b of M.anguillicaudatus has some distinct domains:Pfam domain、transmembrane region、coil domain、RL11（Ribosomal protein L11/L12）and 6-phosphogluconate dehydrogenase,C-terminal domain.Using hi TAIL-PCR method,a 1800 bp upstream of the transcription start site（TSS）of the cyp19a1b gene was obtained and defined as promoter sequence.Meanwhile,the bioinformatics analysis for sequence showed that there were some common transcription factor binding sites in cyp19a1b gene,including Ftz、SRF、SP1、GR、PR、Myo D、SOX-9、SOX-4、E₂、ER and et al.2.Difference analysis of cyp19a1 between sex and ploidy of M.anguillicaudatus1)Taking diploid loach as the object,the brain,gonad,liver,muscle and plasma tissues of four typical gonadal development stages were collected to compare the expression differences of cyp19a1 gene between sex in different developmental stages.The results showed that the expression level of cyp19a1a in ovary was the highest and significantly higher than that in testis（P<0.05）,while the expression level in brain was lower.The expression level of cyp19a1a was higher in stage II and III of ovarian development,but decreased sharply in stage IV and reached the lowest level in stage V.In testis,the expression level of cyp19a1a decreased with gonad development.The expression level of cyp19a1b in brain was significantly higher than that in gonad（P<0.05）,and the expression level in female brain was higher than that in male（P<0.05）;the expression level of cyp19a1b in brain was lower in stage II and III,but increased significantly in stage IV and V（P<0.05）;the expression level of cyp19a1b in testis was higher than that in ovary in all developmental stages.2)The changes of plasma estradiol（E₂）and testosterone（T）in loach were related to their developmental stages.The plasma E₂level was the highest in the third stage of gonadal development,and the plasma E₂level of female loach was always higher than that of male loach;the plasma T concentration of female and male loach increased first and then decreased,with the highest in stage IV,and the plasma T concentration of female loach was always lower than that of male loach.In addition,the changes of E₂and T were consistent with the expression of cyp19a1a and cyp19a1b in gonad.3)The expression of cyp19a1 between-tissues and-ploidy of 12-month-old diploid and tetraploid loach was analyzed.The results showed that the expression of cyp19a1a in all tissues of tetraploid was higher than that in diploid,excepted in testis;in female loach,the expression level of cyp19a1b in brain of tetraploid was significantly higher than that in diploid,but the expression level in male was significantly lower than that in diploid（P<0.05）.Combined the results obtained in the present study,we suggest that the relatively high expression of cyp19a1a in tetraploid may be related to the development stage of gonad,and later sexual maturity is conducive to more energy for individual growth.3.Correlation between the DNA methylation and gene expression of cyp19a11)In every stages of gonadal development,cyp19a1a gene was always hypermethylated in testis,and the average methylation level in testis was higher than that in ovary;with the development of gonad,the methylation level of cyp19a1a in ovary decreased first and then increased.Correlation analysis showed that there was a significant negative correlation between the expression level of cyp19a1a gene and the methylation level（R²=0.81）,indicating that DNA methylation was involved in the regulation of cyp19a1a expression in gonads,and then regulated the sex differentiation and gonadal development of loach.2)The average DNA methylation level of cyp19a1b gene in loach had no significant difference between male and female,and there was no significant change in the four stages,with the values ranging from 60%to 80%.Correlation analysis showed that the average methylation level of cyp19a1b gene was not correlated with gene expression（R²=0.19）.3)DNA methylation is catalysed and maintained by DNA methyltransferases（dnmts）.The above results showed that the expression level of cyp19a1a was negatively correlated with its methylation.This part further analyzed the correlation between the expression level of cyp19a1a and dnmts.The results showed that there was no significant correlation between the expression level of dnmt1,dnmt3aa and dnmt3b and the expression level of cyp19a1a,but the expression level of dnmt3ab and cyp19a1a was significantly correlated There was a negative correlation（r=-0.535,P=0.007）and a significant positive correlation（r=0.773,P=0.025）with DNA methylation level of cyp19a1a.It is speculated that dnmt3ab may regulate the expression of cyp19a1a gene by affecting the methylation level of cyp19a1a in gonad.The correlation analysis between the expression level of dnmts and the expression level of cyp19a1b showed that there was no significant correlation between dnmts gene and the expression level of cyp19a1b,but there was a significant positive correlation between dnmt3b and the DNA methylation level of cyp19a1b（r=0.899,P=0.002）.