CAJ | 학술논문

用Nd:YAG激光微束将处于丝分裂中期的小麦(Triticum aestivum L.)6B染色体微切割为四段,并用微细玻璃针将每个片段分别回收.将分离的染色体片段DNA用Sau3A接头介导的多聚酶链式反应(LA-PCR)分别扩增.Southem杂交证明4个特定区域的DNA确实来自于小麦基因组.用一系列(42对引物)位于6B染色体上的微卫星序列对微切割的染色体片段的PCR产物进行了验证.结果表明,获得的染色体片段的PCR产物来自于小麦6B染色体.将6B染色体4个片段的第二轮PCR产物克隆到pGET-vector中,建立了4个染色体特定区域的基因组文库,命名为R1、R2、R3和R4,分别包含2.1×105、2.74×105和2.93×105个重组子克隆.每个文库均随机挑选150个克隆进行质粒的小量制备和酶切验证.结果显示;插入片段大小在300～1800之间,平均大小为820～870bp,其中43%～48%的克隆为低/单拷贝序列,42%～47%为中/高拷贝序列.本研究为详细分析植物单染色体的不同片段的分子遗传学研究提供了基础.
용Nd:YAG격광미속장처우사분렬중기적소맥(Triticum aestivum L.)6B염색체미절할위사단,병용미세파리침장매개편단분별회수.장분리적염색체편단DNA용Sau3A접두개도적다취매련식반응(LA-PCR)분별확증.Southem잡교증명4개특정구역적DNA학실래자우소맥기인조.용일계렬(42대인물)위우6B염색체상적미위성서렬대미절할적염색체편단적PCR산물진행료험증.결과표명,획득적염색체편단적PCR산물래자우소맥6B염색체.장6B염색체4개편단적제이륜PCR산물극륭도pGET-vector중,건립료4개염색체특정구역적기인조문고,명명위R1、R2、R3화R4,분별포함2.1×105、2.74×105화2.93×105개중조자극륭.매개문고균수궤도선150개극륭진행질립적소량제비화매절험증.결과현시;삽입편단대소재300～1800지간,평균대소위820～870bp,기중43%～48%적극륭위저/단고패서렬,42%～47%위중/고고패서렬.본연구위상세분석식물단염색체적불동편단적분자유전학연구제공료기출.
Chromosome 6B of wheat (Triticum aestivum L.) in mitotic metaphase spreads was micro-dissected with Nd:YAG laser microbeam into four segments and then each segment was collected by glass needles. The DNAs of the isolated chromosomal segments were separately amplified by Sau3A linker adaptor-mediated polymerase chain reaction (LA-PCR). The presence of region-specific DNA from each of four segments was verified by Southern hybridization. The second round PCR products from four segments of chromosome 6B were cloned into a pGEM T-vector to create four chromosome regionspecific libraries, named R1, R2, R3 and R4, which included 2.1×105; 2.74×105; 2.45×105 and 2.93×105recombinant clones, respectively. A total of 150 randomly selected clones from each library were characterized by mini plasmid DNA preparation and enzyme restriction. Results showed that the size of inserts ranged from 300 to 1 800 bp with an average of 820 to 870 bp, of which 43%-48% were low/unique copy and 42%-47% were medium/high copy sequences. A set of microsatellite sequences located on chromosome 6B and other chromosomes of wheat were used for the verification of PCR products from micro-dissected chromosomal segments. The results reported here should facilitate the molecular genetics analysis of different fragments from single chromosomes of a plant.