玉米自交系齐319细菌人工染色体文库的构建及鉴定  

Construction and Characterization of a Bacterial Artifcial Chromosome Library of Maize(Zea mays L.) Inbred Line Qi319

作　　者：穆春华[1,2,3] 张发军[1,2,3] 杨煜[1,2,3] 李文才[1,2,3] 鲁守平[1,2,3] 孙琦[1,2,3] 于彦丽[1,2,3] 孟昭东[1,2,3] 张秀清[1,2,3] 李广存[1,2,3] 

机构地区：[1]山东省农业科学院玉米研究所 [2]山东省农业科学院蔬菜研究所 [3]山东省作物遗传改良与生态生理重点实验室,济南250100

出　　处：《农业生物技术学报》2013年第11期1389-1395,共7页Journal of Agricultural Biotechnology

基　　金：山东省自然(青年)科学基金项目(No.ZR2012CQ033);农业部转基因重大专项(No.2008ZX08003-001)

摘　　要：玉米(Zea mays L.)病害的发生和流行一直是影响其生产的限制因素,玉米南方锈病、粗缩病是我国东南玉米区及黄淮海夏玉米区的重要病害。尽管目前已经开展了对两种病害抗性基因的遗传规律、染色体定位等相关研究,但对这些基因的克隆及功能鉴定等研究进展缓慢,构建抗性材料BAC文库并对抗病基因进行克隆是目前较为有效的方法之一。本研究以黄淮海夏玉米区优良抗病玉米自交系齐319为材料,利用CopyControl pCC1为载体,通过高分子量DNA的提取、部分酶切与大片段DNA的选择、连接转化等多个方面的优化,构建了玉米细菌人工染色体文库。该文库包含270 720个克隆,平均插入片段大小约90 kb,空载率为1.3%,约覆盖玉米基因组10.43倍。文库构建为这些重要功能基因的克隆及比较基因组学研究提供了基础资料。Maize（Zea mays L.） is the crop that has the highest area and yield in China since 2012. The incidence and prevalence of corn diseases affecting maize yield, especially southern corn rust and rough dwarf disease which broadly distribute southeast of China and Huanghuaihai maize zone, cause great losses. It is important to use the resistant germplasms for maize breeding. The bacterial arti？cial chromosome （BAC） cloning system is an invaluable tool in the cloning of disease resistance genes and in conducting structural and functional analyses. A BAC library for Qi319, the key source for disease-resistant maize breeding in China, was constructed in this study. Based on the modifications consisted of using etiolated cotyledon from 7 to 9 d growing plants as the DNA preparation source and addition of PVP-40 and β-mercaptoethanol in the extraction-washing buffer, we used 100 to 200 kb partial digested DNA fragments for 233 transformations. The fragments were ligated into CopyControl pCC1. The library contains 270 720 clones with an average insert size of 90 kb. Up to 1.33% of clones had no insert. Based on a haploid genome size of 2 300 Mb, the coverage of the library is about 10.43 genome equivalents, providing 99.99% possibility to isolate any maize gene or sequence in the library. No visible changes from the EcoRⅠ restriction patterns of four BAC clones between 20 and 100 generations indicated the stability of the BACs. In the future, the BAC library will serve as both a giant gene resource and an invaluable tool for map-based gene isolation, physical mapping and comparative genome analysis.

关 键 词：玉米 齐319 抗病基因 细菌人工染色体文库 

分 类 号：S513[农业科学—作物学]