性类固醇激素在菲律宾蛤仔性逆转中的作用及分子机制研究

Sex reversal is widespread in bivalves as a kind of important reproductive regulation phenomenon. The overall sex reversal ratio of Manila clam Ruditapes philippenarum was 19.0% in the natural population. This provides an effect approach for establishing a large scale of self-inbreeding families. Sex steroid plays complicate effects in regulation of shellfish sex differentiation, even surpasses genetic determination. In this project, the effects of sex steroid on the sex reversal of Manila clam will be conducted using LC-MS/MS, RNA-seq, qRT-PCR. The aim is to analyze molecular Synthetic metabolic pathways of the endogenous steroids in the process of sex reversal; illuminate molecular regulation mechanism of exogenous steroids on the sex reversal;explore the miRNA associated with sex reversal;discuss the sex reversal genetic mechanism; establish the correlation regulation model exogenous steroids-sex reversal gene-endogenous steroids for Manila clam; develop an effect technology to induce the sex reversal, and establish a large scale of self-inbreeding.This project could enrich and complementary theories of the function of sex steroids on sex determination and differentiation of molluscs, moreover, the establishments of inbred lines should promote the research on the Manila clam in genetic breeding, genome mapping, shell color, shell morphology genetic mechanism.

性逆转作为一种重要的生殖调控现象，在双壳贝类中广泛存在。在天然群体中约19.0%菲律宾蛤仔发生性逆转，这为解决菲律宾蛤仔大规模自交系构建的难题提供了有效途径。性类固醇激素对贝类性别分化的调控过程最为复杂，甚至超过遗传因素的作用。本项目将采用LC-MS/MS、RNA-seq、qRT-PCR等技术开展性类固醇激素对菲律宾蛤仔性逆转作用分子机制研究，解析内源性类固醇激素在性逆转过程中分子合成代谢途径；阐明外源性类固醇激素对性逆转分子调控机制；探讨性逆转遗传机制；探索miRNA与性逆转相关靶基因调控关联机制；建立菲律宾蛤仔外源性类固醇激素-性逆转基因-内源性类固醇激素关联调控模型。在此基础上，开发菲律宾蛤仔性逆转高效诱导技术，大规模构建自交系。本项目的开展可以丰富和补充软体动物性别决定和分化理论基础，同时自交系的构建对菲律宾蛤仔遗传育种、基因组作图、壳色、壳型遗传机制解析等研究具有积极的推动作用。

本项目采用LC-MS/MS、RNA-seq、qRT-PCR等技术开展性类固醇激素对菲律宾蛤仔性逆转作用分子机制研究，结果表明，睾酮500ng/L组、雌酮500ng/L组和雌酮1µg/L组个实验组中分别发现雌雄同体个体。不同激素种类和同种激素不同浓度处理对蛤仔内源激素水平影响差异显著。10μg/L雌二醇诱导蛤仔性逆转效果最好，首次发现了性类固醇激素作用下能够出现雌、雄同体个体；3个Dmrt基因的表达量从担轮幼虫到壳顶幼虫时期迅速增长，其中Dmrt4-like-2在蛤仔的不同发育时期均有表达。推测蛤仔可能从担轮幼虫开始出现性别分化。选取了对照组(E)、雌二醇刺激组（D）和雌雄同体(ET)组进行转录组学分析，与对照组相比激素刺激组有697个差异表达基因，其中288个基因下调、409个基因上调. 雌雄同体组与激素刺激组相比, 获得了495 个差异表达基因, 79 个基因上调 、 419个 基因下调。雌雄同体组与对照组相比，获得了208个差异表达基因, 73个基因上调、105个基因下调，发现了一批与性别决定、性别分化及性逆转相关基因。以上研究为探究蛤仔性逆转分子调控机制、研发菲律宾蛤仔性逆转高效诱导技术奠定了理论基础。