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以四倍体马铃薯栽培品种‘合作88’培养12~15 d苗龄的幼嫩叶片为试材,提取高分子量基因组DNA,利用限制性内切酶HindⅢ部分酶切后回收100~300 kb片段,连接到载体Copy ControlTM p CC1BACTM的HindⅢ位点上,构建了细菌人工染色体(BAC)文库。该文库约由850 000个克隆组成,空载率约2.08%,保存在1 700个混合池中,克隆插入片段平均大小约为90 kb,覆盖单倍型马铃薯基因组约85倍。随机挑取6个BAC克隆连续培养5 d,提取DNA进行HindⅢ完全酶切检测,确认不同培养时间的BAC克隆的指纹图谱完全一致,表明文库较好的稳定性。BAC文库的构建为马铃薯重要农艺性状基因克隆、基因组测序以及比较基因组研究等奠定了基础。
The tetraploid potato cultivar ’Cooperative 88’ was used to cultivate young leaves of 12-15 days old seedlings as test material, high molecular weight genomic DNA was extracted, partially digested with restriction endonuclease Hind Ⅲ to recover 100-300 kb fragments, Was ligated to the HindIII site of the vector Copy ControlTM p CC1BACTM to construct a bacterial artificial chromosome (BAC) library. The library, composed of about 850,000 clones with an empty-loading rate of about 2.08%, was stored in 1,700 pools. The average size of the cloned insert was about 90 kb, covering about 85-fold of the haplotype potato genome. Six BAC clones were randomly selected and cultured continuously for 5 days. The DNA was extracted and subjected to complete HindIII digestion. The fingerprints of BAC clones at different incubation times were identical, indicating a good stability of the library. The construction of BAC library lays the foundation for gene cloning, genome sequencing and comparative genomics of important agronomic characters in potato.