高粱无融合生殖基因的定位、分离与分析

项目名称： 高粱无融合生殖基因的定位、分离与分析

项目编号： No.31470285

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 张春来

作者单位： 山西省农业科学院高粱研究所

项目金额： 85万元

中文摘要： 无融合生殖具有固定杂种优势的潜力，是植物生殖生物学的重要研究领域。高粱可做为禾谷类作物无融合生殖研究的模式植物，但其分子机制却知之甚少。 以本所选育、国际领先的高频率无融合生殖系2083×常规系E35B杂交F2分离群体及F5代无融合/有性生殖近等基因系为试材, 精细定位控制无融合生殖的位点在染色体区段上。对大孢子母细胞期、减数分裂期等胚珠发育过程的转录组用Solexa Hiseq法作深度测序，比较和筛选出无融合相关的差异表达基因，再根据已公开的全基因组序列，用透射法定位到所获无融合生殖的染色体区段上，得到控制无融合生殖的基因。对基因进行分类并做荧光定量RT-PCR表达分析，克隆和构建转基因植株，分析超量和抑制表达下无融合生殖基因功能。DNA测序和序列列阵比较，分析源于高频无融合生殖系和常规系等位基因DNA多态性，并对多来源无融合生殖系做DNA鉴定,建立分子标记以辅助选育更优系。

中文关键词： 无融合生殖；分子机制；基因表达分析；基因定位与分离；高粱

英文摘要： Apomixis is the most active area of plant reproductive biology research as it allows fixing heterosis after only hybridization once. Sorghum is the model species of cereal apomixis research with its molecular mechanism is still barely understood. We are pioneering in the development of sorghum breeding line 2083 with high frequencies of apomixis and producing advanced experimental hybrids capable of fixing heterosis. This project is designed to fine mapping the apomictic loci using 120 evenly distributed SSR markers on 10 chromosomes and 2083 × E36B F2 segregating population and F5 isogenic inbred lines. Meanwhile, deep sequencing the transcriptome of ovule development by Solexa Hiseq will proceed and unveil the differentially expressed genes in magaspore mother cell, meiosis and mature embryo sac of apomictic ovule development stages by contrasting apomictic and non-apomictic lines.Differentially expressed apomictic candidate genes will be projected to above obtained apomixis chromosome regions based on the publicly available Sorghum near-whole Genome Sequence.The final apomictic gene sets will be catalogued and further analyzed by real-time reverse transcript PCR and compared to canonical apomictic genes such as Agonaute, Apostart and DDM1. The apomictic genes will be cloned into Gateway vectors and functionally characterized in transgenic plants by overexpression and ds-RNA suppression. Genomic DNA will be cloned by PCR and analyzed for allelic variations of homologues between apomictic and normal lines by multi-sequence alignment. Screening diverse apomictic lines and conventional lines will be carried out using the derived DNA markers. In the future it may be possible to transfer a few of these genes into normal sorghum lines to induce apomixis at a much higher frequency.

英文关键词： apomixis;molecular mechanism;gene expression analysis;gene mapping and isolation;sorghum