高抗黑胫病烤烟BAC文库的构建及分析

Construction and characterization of a BAC library for flue-cured tobacco line with high resistance to blank shank

烟草是重要的模式植物。本研究利用pIndigoBAC536-S载体及Hind Ⅲ限制性内切酶酶解烟草基因组DNA的方法,构建了烟草新品系14-60的细菌人工染色体(BAC)文库。该文库共包含414,720个克隆,保存在1080块384板中。随机挑选的120个烟草BAC克隆检测结果表明,外源插入片段大小为97.0~145.5 kb,平均约为123 kb,空载率极低(0),覆盖烟草基因组11倍。用烟草hem A基因、eIF4E-1基因、NtFT基因的特异引物进一步验证,该文库质量高、可用性强,为烟草黑胫病抗性基因的克隆以及其他重要农艺性状和品质性状等功能基因克隆研究提供了基础资源。

Tobacco(N.tabacum)is an important model crop in molecular biology research.In this study,a bacterial artificial chromosome(BAC)library of a flue-cured tobacco line 14–60 with high blank shank resistance and good quality was constructed.High molecular weight DNA was isolated using intact nuclei from tobacco,partially cleaved with Hind Ⅲ and cloned into the pIndigoBAC536-S vector.The BAC library consisted of 414,720 clones arrayed in one thousand and eighty 384-microtite plates,with an average insert size of 123 kb ranging from 97.0–145.5 kb.No empty insert clone was found.Based on an estimated genome size of 4500 Mb for common tobacco,the BAC library was estimated to cover 11 times of genome equivalents.The utility of the library was further confirmed by screening the library with the primers of tobacco hem A,NtFT,and eIF4 E-1 genes.The high capacity library will serve as a giant genomic resource for map-based cloning of quantitative trait loci or genes associated with important agronomic and smoking quality traits or resistance to blank shank,physical mapping and comparative genome analysis.