长牡蛎Htatip2的表达及其在不育三倍体雌性中的DNA甲基化

Expression analysis of Htatip2 and its DNA methylation in sterile triploid females of the Pacific oyster(Crassostrea gigas)

为探究DNA甲基化在长牡蛎性腺发育中的表观遗传调控机制,对长牡蛎Htatip2的同源性、系统进化、组织表达以及三倍体性腺可育型和不育型不同发育时期的基因表达和DNA甲基化谱进行了研究。结果显示,长牡蛎Htatip2的保守结构域与美洲牡蛎的Htatip2-like的保守结构域同源性最高。qPCR分析显示,Htatip2在各个组织中均有表达,其中在雌性性腺中的表达量最高。此外,该基因的表达量在可育型三倍体牡蛎性腺中随着性腺发育成熟而升高,在不育型三倍体牡蛎性腺中表达量变化不显著。BS-PCR分析显示,该基因的甲基化水平随着性腺发育成熟而降低,与基因表达水平成负相关性。双荧光素酶报告结果显示,甲基化的Htatip2启动子片段与未甲基化的片段相比,显著抑制了荧光素酶的活性。研究表明,长牡蛎Htatip2的DNA甲基化可能通过抑制基因表达参与了性腺成熟调控。本研究为表观遗传调控机制参与牡蛎性腺发育提供了重要参考依据。

Sexual maturation in triploid Pacific oysters(Crassostrea gigas)varies widely among individuals.In addition to genetic factors,the influence of the environment on their gonadal development is also crucial.To investigate the epigenetic regulation mechanism of Htatip2 DNA methylation in gonadal development in the Pacific oyster,the homology,phylogeny,gene expression and DNA methylation profiles at different developmental stages of fertile and sterile gonads in the oyster were investigated.The results showed that the conserved structural domain of Htatip2 in the Pacific oyster had the highest homology with the Htatip2-like conserved structural domain in the Eastern oyster(C.virginica).qPCR analysis showed that Htatip2 was expressed in various tissues,with the highest expression in the female gonads.In addition,the expression of Htatip2 increased with gonadal maturation in fertile oysters and did not change significantly in sterile oysters.BS-PCR analysis showed that the DNA methylation level of Htatip2 in oysters decreased with gonadal maturation and was negatively correlated with the gene’s expression level.Finally,the results of dual luciferase reporter assay indicated that the methylated Htatip2 promoter fragment significantly inhibited luciferase activity compared to the unmethylated fragment.This study suggests that DNA methylation of Htatip2 may be involved in the gonadal maturation through inhibition of gene expression.This study provides an important reference for the involvement of epigenetic regulatory mechanisms in gonadal development.