罗氏沼虾性别决定基因的染色体步查与功能鉴定

The molecular regulatory mechanism involved in sex determination remained unknown in the prawns (shrimps) and crabs. No sex determination gene and sex chromosomes have been identified so far. The freshwater giant prawn Macrobrachium rosenbergii is one of the most important cultured species in the wold aquaculture production. The female and male individuals exhibit sexual dimorphic growth patterns, in which males grow faster and reach a larger size at harvest than females. Obviously, all male prawn cultivation will largely increase prawn production and comercial benifit. In this project, we will employ the giant prawn as an experimental animal model. To isolate sex linkage and sex-specfic markers, a high density linkage map will firstly be developed using genome complexity reduction techniques, ect. restriction site-associated DNA sequencing (RAD-seq). And then based on these markers and the ZW chromosomal DNA fragments isolated previously in our lab, positive BAC clones will be screened from the prawn genome library using chromosomal walking strategy, and the sex determination candidate genes are expected to be identified after Illumina high throughout sequencing and annotation. The expression patern of the candidate genes will be examined during sex differentiation/determination.Finally, the functional role of the candidate genes will be characterized with RNAi method to providie insigh into the sex determination mechanism. The output of this project will not only better our understanding on the structure of sex chromsomes and sex evolution of the prawns and crabs, but will provide more useful sex-specific markers for the application in the sex control of the prawn aquaculture industry.

虾蟹类甲壳动物性别决定分子机制仍然是未解之谜, 至今未找到性别决定主效基因和鉴别出性别染色体。罗氏沼虾是世界上最重要淡水养殖虾类之一，雌雄个体的生长速度存在显著差异，性成熟雄虾个体明显大于雌虾，若实现全雄养殖，必将大幅提高养殖产量和经济效益。本研究以罗氏沼虾为实验对象，利用简化基因组技术构建高密度遗传连锁图，筛选性别连锁标记和性别特异分子标记，利用这些标记和我们之前已获得的ZW染色体片段DNA，通过染色体步查筛选性染色体BAC克隆，荧光染色体原位杂交（FISH）方法鉴别性染色体，高通量测序性染色体BAC克隆，发掘性别决定候选基因并解析其结构，分析候选基因在性别决定/分化过程中的表达特征，RNAi方法验证其功能，解析性别决定机制，不仅对于理解虾蟹类性染色体结构、性别遗传的进化具有重要学术价值，而且开发的性别特异分子标记可直接用于罗氏沼虾性别控制生产实践，具有重要应用价值。

虾蟹类甲壳动物性别决定分子机制仍然是未解之谜, 至今未找到性别决定主效基因和鉴别出性别染色体。罗氏沼虾是世界上最重要淡水养殖虾类之一，雌雄个体的生长速度存在显著差异，性成熟雄虾个体明显大于雌虾，若实现全雄养殖，必将大幅提高养殖产量和经济效益。本研究以罗氏沼虾为实验对象，利用简化基因组技术构建高密度遗传连锁图，雌雄整合图谱共包含10,881个SNP标记，覆盖了60个连锁群，总图距为3662.47cM，平均图距为0.34cM。119个连锁标记被定位在21号连锁群上，并且进一步筛选获得11个性别特异分子标记，利用这些标记通过染色体步查筛选ZW性染色体BAC克隆12个，荧光染色体原位杂交（FISH）方法鉴别性染色体，高通量测序性染色体BAC克隆，联合全基因组序列分析，发掘获得Dsx、Fem-1等性别决定候选基因，Dsx只在雄性精巢与促雄腺及雄性幼体特异表达，Fem-1只在雌性卵巢表达，但在雌雄幼体都不表达，RNAi 干扰敲降Dsx基因表达同时也能够抑制促雄腺激素基因IAG表达，持续干扰雄性幼体2个月后获得性反转假雌（ZZ）个体，表明Dsx通过调控IAG基因的表达控制罗氏沼虾雄性性别发育过程，假雌个体（ZZ）与正常雄性（ZZ）个体交配获得全雄子代，该研究结果可为生产上罗氏沼虾性别控制单性化养殖提供新的技术途径，同时为进一步阐明罗氏沼虾性别决定分子机制奠定基础。