普通小麦5DL染色体分选及染色体BIBAC文库构建技术体系优化

 【目的】优化小麦染色体流式分选及特定染色体BAC文库构建技术体系，为加速小麦基因组学研究提供技术支撑。【方法】采用双阻断法对中国春5DL端体根尖分生组织进行细胞周期同步化处理，结合机械匀浆法制备染色体悬浮液进行流式核型分析，分选染色体5DL及PCR检测，对小麦染色体分选技术体系进行优化。对复合峰分选产物Bam HI酶切制备高分子量（high molecular weight，HMW）DNA，按10﹕1质量比与双元细菌人工染色体（binary-bacterial artificial chromosome，BIBAC）连接，电激转化感受态细胞DH10B，随机挑取100个重组子，经NotI完全酶切后脉冲电泳检测插入片段大小，优化小麦染色体BAC文库构建的技术体系。【结果】经1.25 mmol?L-1 羟基脲（hydroxyurea，HU）18 h，恢复6 h和1 µmol?L-1 氟乐灵（trifluralin）2.5 h的顺序处理的小麦根尖分生组织，细胞有丝分裂指数可超过60%。流式核型分析表明，5DL端体的流式核型图由清晰的三个复合峰和两个单峰组成。PCR鉴定表明，其中一单峰确由5DL染色体组成。部分酶切复合峰染色体3～10 min，可以获得50～300 kb 的HMW DNA。连接产物检测显示平均插入片段可达100 kb左右，根据荧光通道值，估算5DL端体的大小约为482 Mb，构建库容为19 000个克隆的5DL端体特异文库即可达到约4倍的覆盖率。【结论】上述两项优化技术适用于小麦染色体的分选及染色体特异文库的构建。

Optimizing Protocol of Flow Sorting Chromosome 5DL(Triticum aestivum L.)and Chromosome-specific BIBAC Library Construction

【Objective】The study aims to optimize chromosome sorting and construction of chromosome-specific library to simplify the wheat global genome research.【Method】Using double-block method to synchronize root tip meristem cells of common wheat ditelosomic line 5DL, the chromosomes were released for flow karyotypic analysis after fixed in 2% polyformaldehyde in Tris-HCl buffer followed by mechanical homogenization. Flow sorting was performed on chromosomes from the putative single peak and composite peaks. PCR method detected flow sorted chromosome 5DL and chromosomes from composite peaks were subjected to 1 U BamHI digestion. In the ligation reaction, the ratio, 10:1 (high-molecular-weight (HMW) DNA:BIBAC vector, w/w) was applied and then 1:20 (ligation:electro-competent cell, v/v) performed in electroporation. Random 100 recombinators were selected for full digestion with NotI to determine the insert sizes.【Result】The mitotic index can reach over 60% after the following treatment with 1.25 mmol?L-1 hydroxyurea for 18 h, 6 h in hydroxyurea (HU)-free Hoagland’s solution for recovery and 2.5-h 1 µmol?L-1 trifluralin. Based on fluorescence microscope and PCR detection, histograms of fluorescence intensity acquired after the analysis of DAPI-stained common wheat ditelosomic line 5DL chromosomes comprised three composite peaks and two single peaks representing chromosome 5DL and 3B, respectively. Partial enzyme digestion of high-molecular-weight (HMW) DNA demonstrated that 50～300 kb fragments can be acquired with an average insert size of 100 kb or so.【Conclusion】The study presents a useful method in constructing chromosome- and chromosome arm-specific library.