中间偃麦草2Ai-2染色体DNA文库构建及特异性序列的克隆

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-specific DNA Sequences

以二体异附加系Z2与其普通小麦亲本宛7107杂交F1的花粉母细胞为材料,用原位杂交方法确定其中的单价体为中间偃麦草2Ai-2染色体;在此基础上,以显微分离、回收、LA-PCR (Linker-adaptor PCR) 扩增了该条染色体,扩增产物长度在0.15～3 kb之间,主要分布在0.2～2 kb。以α-32P-dCTP标记的中间偃麦草的基因组DNA为探针,对LA-PCR扩增产物进行杂交,证实扩增产物来自中间偃麦草。将扩增片段纯化后连接到质粒载体pUC18上,构建了2Ai-2染色体DNA文库。该文库包含约5×105个克隆。随机选取500个克隆进行分析,发现插入片段长200～1 500 bp,平均580 bp。点杂交结果表明,56%为低/单拷贝序列,44%为重复序列。从文库筛选到4个中间偃麦草克隆。RFLP结果表明,3个为多态性低拷贝(Mag065、Mag088、Mag139),1个为高度重复序列克隆(Mag104)。GISH结果表明,Mag104为中间偃麦草染色体专化重复序列。

The univalent from the meiosis-metaphase spreads of F1 (Z2 ×wheat variety Wan7107) was identified to beAgropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes were microisolated and collected. After tworounds of PCR amplification, the PCR products were ranged from 150-3 000 bp, with predominant fragments at about 200-2 000 bp. Using Ag. intermedium genomic DNA as probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformed into competenceEscherichia coli DH5α to produce a 2Ai-2 chromosome DNA library. The microcloning experiments produced approximately5 105 clones, the size range of the cloned inserts was 200 -1 500 bp, with an average of 580 bp. Using Ag. intermediumgenomic DNA as probe, dot blotting results showed that 56% clones are unique/low copy sequences, 44% are repetitivesequences in the library. Four Ag. intermedium clones were screened from the library by RFLP, and three clones (Mag065,Mag088, Mag139) belong to low/single sequences, one clone (Mag104) was repetitive sequence. GISH results indicatedthat Mag104 was Ag. intermedium species-specific repetitive DNA sequence.