玉米抗粗缩病基因qMrdd2的克隆与功能分析

Maize is a staple food, feed crop and industrial raw materials. Rough dwarf disease is one of the main diseases in Huang Huai summer maize production area in China. Increasing the resistance of varieties is an economical and effective way to control the damage of this disease. With high density genetic map of RIL population derived from a cross between the inbred lines Qi319 and Ye478, the qMrdd2 was located on the chromosome 2, with a physical distance of 0.15Mb between bin-markers Mk807 and Mk808. Then, qMrdd2 was narrowed into in the physical distance of 2Mb with the mapping population, derived from the cross between chromosome segment substitution line with the genetic background of Ye478, donor of Qi319, and background parent. In this study, the qMrdd2 will be fine mapped into the range of 100kb by screening and identification of the recombinants, this resistance gene for qMrdd2 will be map-based cloned by sequence of the BAC clone from Qi319 library. Using the vectors of genetic complementation vector, gene knockout and over expression, the function of qMrdd2 gene will be verified. By analyzing the gene expression pattern, subcellular localization and interaction genes of qMrdd2, the molecular mechanism and regulation pathway of maize resistance to rough dwarf virus will be analyzed.

玉米是重要的粮食、饲料作物和工业原料。粗缩病是我国黄淮海夏玉米生产区的主要病害之一，提高品种抗性是控制该病危害的有效措施之一。利用抗粗缩病自交系齐319与感病材料掖478构建的RIL群体，结合高密度遗传图谱将抗粗缩病基因qMrdd2定位在第2染色体bin标记Mk807-Mk808之间的物理距离为0.15Mb的范围内。在此基础上，利用本实验室构建的以掖478为遗传背景、齐319为供体的染色体片段置换系群体及次级精细定位群体，将qMrdd2锚定在2Mb的范围内。本项目拟通过重组单株的筛选及后代表型鉴定将qMrdd2精细定位在100 kb范围内，结合齐319 的BAC文库分析候选基因结构，对抗性基因进行图位克隆。利用遗传互补载体、基因敲除载体及过表达载体的转基因试验，验证候选基因功能。通过qMrdd2基因表达特性分析、亚细胞定位及互作基因验证等分子生物学方法，解析玉米抗粗缩病的分子机制及调控途径

玉米是重要的粮食、饲料作物和工业原料。粗缩病是我国黄淮海夏玉米生产区的主要病害之一，提高品种抗性是控制该病害的有效措施之一。项目前期利用抗粗缩病自交系齐319与感病材料掖478构建的RIL群体，结合高密度遗传图谱将抗粗缩病基因qMrdd2定位在第2染色体bin标记Mk807-Mk808之间的物理距离为0.15Mb的范围内。本项目，我们通过精细定位群体将qMrdd2定位于标记RD81和RD87之间物理距离为315 Kb的区间内（B73 RefGen_V3）,关联分析结果表明qMrdd2候选抗病基因ZmGLK36启动子区域的26 bp的插入/缺失与病情指数显著相关。ZmGLK36的过表达、互补和基因敲除株系人工接种鉴定表明，ZmGLK36为qMrdd2的抗玉米粗缩病基因。ZmGLK36定位于细胞核内，qRT-PCR结果表明其表达量升高可以抑制RBSDV的积累。酵母双杂交试验表明，ZmGLK36与RBSDV 13个ORF编码的蛋白没有互作；转录组学测序和代谢组分析表明，ZmGLK36主要参与植物激素信号途径来实现对RBSDV的抑制，其中包括茉莉酸JA和GA途径。qRT-PCR、酵母单杂交与双荧光素酶试验表明，ZmGLK36受病毒诱导后激活下游ZmJMT基因的表达，参与茉莉酸合成途径。综上所述，本项目研究结果将为进一步解析ZmGLK36精准调控JA与GA途径，实现对RBSDV的抑制和缓解玉米生物量的降低奠定材料与数据基础。