一种新的家蚕无生殖细胞突变及其分子机制研究

Procreation and breed are the important problems of Sericultural science. A non-germ-cell mutant had been discovered in our lab. In this research, its physiological mechanism will be analyzed by using histological and anatomical observation on the developmental status of the internal genitals and germ cell. The mutant gene ngc will be fine mapped by integrated application of SSR and SNP molecular markers, then the full length cDNA of this gene will be cloned. The transgenic vectors of the wild-type ngc gene will be constructed, and will be transferred into a new silkworm strain. The transgenic line which the heterogenous wildtype ngc would be proved to be merged into a chromosome different from the chromosome which the mutant located will be used to cross to the mutant line. The function of the gene will be proved by the recover of the reproductive capacity in the transgenic line which is the homozygous in the mutant locus. The marker-selective techniques will be constructed based on the difference of genomic sequence in the ngc site between the wild type and mutant. The model of the gene action and its molecular mechanism will be analyzed by research on the differences of proliferation, differentiation, development of germline cells and gonad cells, and the gene expression profile of cell differentiation related signal pathways in the gonad between the wild type and mutant. The research results will be useful in revealing the molecular mechanism for the development regulation in silkworm germ cell, and will be useful in finding the target genes for controlling the pest population based on the inherited reproductive disorders.

生殖与繁育是蚕业科学的重要领域。本项目以本实验室新发现的家蚕生殖细胞发生发育突变- - 无生殖细胞（non-germ-cell）突变系统为主要对象，从组织解剖学角度研究突变体家蚕内生殖器官和生殖细胞的发育状况，分析其生殖障碍发生的生理机制。综合应用SSR和SNP分子标记定位技术和RACE技术，精细定位并克隆无生殖细胞突变基因（ngc）。构建其野生型基因转基因载体，建立异位表达野生型基因的转基因品系，杂交导入突变品系，以突变体恢复正常生殖能力为指标验证该基因功能。以突变型和野生型的ngc基因位点序列差异为依据，建立两种基因型个体的分子标记鉴定选择技术，研究野生型与突变体的生殖系细胞和生殖腺细胞的增殖分化发育的差异、细胞分化相关信号通路基因的表达差异，明确该突变基因的作用模式及分子机制。研究结果将有助于揭示家蚕生殖细胞发育调控的分子机制，为利用生殖障碍进行害虫种群数量的遗传控制提供靶标基因信息。

研究背景：鳞翅目害虫占世界农业害虫的80%左右，家蚕是鳞翅目模式昆虫，从家蚕的突变资源出发，筛选出影响鳞翅目昆虫生长发育、繁殖等生命过程的关键基因，明确其分子机制，对害虫防治新技术、新途径的开发有重大的意义。.主要研究内容：针对本团队新发现的一个家蚕生殖障碍突变系统-家蚕无生殖细胞突变（mutation of non-germ-cell，ngc），从遗传学、解剖学、突变基因定位、候选基因克隆与表达分析等方面开展研究，力图明确其遗传、生理与分子机制。. 重要结果：（1）家蚕无生殖细胞突变性状呈假母性遗传，受1个隐性基因控制，但个体间分离比例有一定波动。（2）突变体雌性个体卵巢管内没有卵子及卵室形成，卵巢管的伸长受到明显抑制，突变体雄性个体精巢小室内没有发现精子束的存在。推断突变型个体的卵子或精子在发生、迁移或成熟阶段可能存在异常。（3）通过对M、LMe等4个不同特点家蚕突变基因的连锁定位分析，建立并优化了利用HRM技术进行突变基因的精细定位技术，将ngc精细定位于家蚕基因组nscaf2865:4957849_4958073和nscaf2865:5096445_5096643之间。（4）克隆该区域内基因，定名为家蚕体轴抑制蛋白基因（Bmaxin）。在突变纯合体(ngc/ngc)与野生型（+ngc/+ngc）之间，野生型与突变型Bmaxin基因在蛋白质编码区内编码的氨基酸序列完全一致。突变纯合体与野生型之间，Bmaxin基因的3’UTR区域的基因组序列虽然基本相同，但mRNA序列表现出差异，突变体mRNA提前出现polyA尾巴、导致3’UTR长度变短373bp，野生型与突变型之间的Bmaxin基因存在着选择性加尾的现象。（5）通过对Bmaxin基因表达量进行qRT-PCR分析，表明突变体的Bmaxin基因表达量低于野生型，Bmaxin基因3’UTR的截短导致了mRNA分子的稳定性改变。（6）axin基因是Wnt/β-catenin信号通路的关键成员，初步推断家蚕无生殖细胞突变的表型出现可能与家蚕Axin蛋白表达量有关、与Wnt信号转导途径有关。.科学意义：首次在家蚕的遗传突变中发现基因的选择性加尾现象，首次发现昆虫的生殖细胞的发生或分化成熟与 Wnt/β-catenin信号通路有关。