| Scapharca broughtonii is an important large-scale benthic shellfish in the Yellow and Bohai Sea areas and an important economic aquaculture shellfish in the north of China.In-depth research on the gonad development characteristics of S.broughtonii can provide support for the successful development of artificial seedling breeding.Shellfish gonadal development is regulated by both endogenous causes,which are related genetic material;and exogenous causes,which include environmental factors such as temperature,salinity,p H,and photoperiod.As the key substances for sex maintenance and gonad development in aquatic organisms,sex steroid hormones play important roles in the regulation of shellfish gonad development,but there is a relative lack of relevant studies on S.broughtonii.In this study,a method for the detection of sex steroid hormones(progesterone,testosterone and estradiol)was established to determine and analyze the variation of the content and proportion of sex steroid hormones in testes and ovaries at different gonad development stages of S.broughtonii.Whole-transcriptome sequencing of the ovaries at three gonad developmental stages of S.broughtonii,and the regulation effects of m RNA and non-coding RNA on gonad development were analyzed.The sequence characteristics and temporal and spatial expression of four genes related to sex steroid hormone synthesis: Steroidgenic factor 1,(SF-1),Steroidogenic acute regulatory protein(St AR),17β-hydroxysteroid dehydrogenase 14(17β-HSD14),Cytochrome P450 17(CYP17),and three circadian clock genes: Brain and muscle ARNT-like protein 1(Bmal1),Cryptochrome(Cry),Period(Per)were further identified and analyzed.The main research contents and results are as follows:1.Distribution characteristics of sex steroid hormones in the gonad developmental cycle of S.broughtoniiThe histological method was used to divide the gonadal development of S.broughtonii into five stages,and the contents of three important sex steroid hormones,progesterone,testosterone and estradiol,in the gonadal development cycle were detected by enzymelinked immunosorption assay(ELISA).The results showed that the contents ranges of progesterone,testosterone,estradiol and total content in ovaries were 157.04 ～ 759.01 pg/g,18.86 ～ 56.03 pg/g,14.80 ～ 57.90 pg/g and 190.70 ～872.94 pg/g,respectively.And the range of progesterone,testosterone,estradiol and total contents in testes were 211.81-470.99 pg/g,23.22-65.17 pg/g,17.52-38.00 pg/g and 252.55-574.16 pg/g,respectively.Progesterone,testosterone and estradiol accounted for 80%,10% and 10% of the total sex steroid hormone content in the gonads of S.broughtonii,respectively.The contents of stage Ⅱ progesterone,testosterone and estradiol in ovaries and testes of S.broughtonii were higher than those in the other four periods,and the total hormone content in stage Ⅱ was also higher than that in the other four periods.The content of progesterone in ovaries except stage Ⅴ were higher than that in testes,the content of testosterone in testes was higher than that in ovaries,the content of estradiol in ovaries of stage Ⅰ,stage Ⅱ and stage Ⅲ was higher than that in testes,and the total content of hormone in the ovary except stage Ⅴ was higher than that in testes,and some of the difference of hormone content between males and females was significant.2.Construction of whole-transcriptome gene regulatory network in ovaries of S.broughtoniiWhole transcriptome sequencing of nine samples of S.broughtonii ovaries were performed by using RNA-seq and small RNA-seq techniques to explore the expression patterns of m RNA and nc RNA in different gonadal developmental stages of S.broughtonii,and key genes and nc RNAs affecting S.broughtonii reproduction were screened at the whole-transcriptome level.The results showed that a total of172.84 Gb Clean reads were obtained after RNA library sequencing,and the Q30 base rate was all greater than 92.06 %.A total of 103.32 M Clean Reads were obtained by small RNA library sequencing,and the Q30 base rate was all greater than 94.57 %.In nine samples,45,453 m RNAs,69,768 lnc RNAs,3810 circRNAs and 1239 mi RNAs were identified.In the three groups,11,342 differentially expressed genes,6897 differentially expressed lnc RNAs,135 differentially expressed circRNAs and 164 differentially expressed mi RNAs were detected.The results of ce RNA analysis of differentially expressed nc RNAs and target genes in S.broughtonii showed a total of101 RNA interactions,including 10 circRNAs,4 mi RNAs and 87 m RNAs.The circRNA-mi RNA-m RNA regulatory network includes six genes associated with gonadal development,providing new insights into the molecular mechanisms of the effects of nc RNA on ovarian development in shellfish.3.Bioinformatics analysis of genes related to sex steroid hormone synthesis and their correlation with gonad developmentThe c DNA sequences of SF-1,St AR,17β-HSD14 and CYP17 genes were obtained from the whole transcriptome of S.broughtonii,and the CDS regions of the genes were sequenced and verified.The CDS sequence of SF-1 gene was 1671 bp,encoding 556 amino acids;the CDS sequence of St AR gene was 1446 bp,encoding481 amino acids;the CDS sequence of 17β-HSD14 gene was 822 bp,encoding 273 amino acids;and the CDS sequence of CYP17 gene is 1527 bp,encoding 508 amino acids.The relative expression levels of four genes related to sex steroid hormone synthesis in ovaries of S.broughtonii were the highest at stage Ⅱ,which was consistent with the highest contents of progesterone,testosterone,estradiol and the total contents of three hormones.In addition,the expression level increased from stage Ⅰ to stage Ⅱ,reached the highest value in stage Ⅱ,then decreased from stage Ⅱ to stage Ⅴ,and reached the lowest value in stage Ⅴ,which was consistent with the trend of the contents and total contents of the three hormones in the process of gonad development.In situ hybridization results showed that positive signals for St AR,17β-HSD14 and CYP17 genes were detected in ovarian mature oocytes,follicular walls and testes spermatogenic cells and sperm,and the positive signal of CYP17 gene in spermatogonia cells was stronger than that in sperm cells.Positive signals for St AR,17β-HSD14 and CYP17 genes were detected in the cytoplasm of the foot,mantle,hepatopancreas muscle and gill of S.broughtonii.4.Spatiotemporal expression characteristics of three circadian clock genes and regulation of related genes in the sex steroid hormone synthesis pathway by Bmal1Three important circadian clock genes,Bmal1,Cry and Per,were identified by whole transcriptional data.The CDS sequence of Bmal1 gene was 2028 bp,encoding675 amino acids;the CDS sequence of Cry gene was 1581 bp,encoding 526 amino acids;and the CDS sequence of Per gene was 4149 bp,encoding 1382 amino acids.Bmal1,Cry and Per were all expressed in the ovarian tissues of S.broughtonii,and their relative expression levels were highest in stage Ⅱ,and the expression levels in stage Ⅱ were significantly higher than those in the other four stages(P < 0.05).The expression of circadian clock genes Bmal1,Cry and Per,and the genes related to sex steroid hormone synthesis SF-1,St AR,17β-HSD14 and CYP17 showed the same change trend during gonad development,which showed the increase and then decrease,and the expression level was the highest in ovary at stage Ⅱ.In situ hybridization results showed that the positive signal of Bmal1 gene was detected in ovarian mature oocyte cells,follicular wall,and testes spermatogonia,sperm,the positive signal of spermatogonia was significantly stronger than that of sperm,indicating that the expression level of Bmal1 gene in spermatogonia was higher than that in sperm,which may play roles in spermatogonia differentiation.Positive signals of Bmal1 were detected in the cytoplasm of foot,mantle,hepatopancreas muscle and gill of S.broughtonii,indicating that the circadian clock gene is widely distributed in the tissue of S.broughtonii and may be involved in the basic life activities.After interfering the expression of the circadian clock gene Bmal1 by using ds RNA technology,the relative expressions of St AR,17β-HSD14 and CYP17 related to the sex steroid hormone synthesis pathways were decreased,suggesting that the circadian clock gene may regulate the synthesis of sex steroid hormones by affecting related genes. |
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