CAJ | 학술논문

采用玻璃针分离法,通过显微操作系统成功地分离到西瓜(2n=22)的第一单染色体,将分离到的西瓜染色体放入0.2 mL Eppendorf管中,经去蛋白,Sau3A酶切,并在染色体DNA片段两端加上Sau3A人工接头后,进行两轮PCR扩增,得到0.4～2.0 kb之间的DNA片段.用西瓜的基因组标记成探针,与单染色体扩增产物进行southem杂交,表明这些西瓜单染色体扩增片段与西瓜基因组DNA之间有同源性,从而证明单染色体DNA确实已被成功地扩增.将第2轮PCR产物构建质粒文库,得到约20 000个重组子.这是染色体微分离、微克隆技术首次在瓜类作物中的应用,该方法为直接从西瓜单条染色体DNA文库中筛选分子标记奠定了基础,说明利用染色体显微分离、克隆技术对西瓜等瓜类作物基因组进行研究是可行的.
채용파리침분리법,통과현미조작계통성공지분리도서과(2n=22)적제일단염색체,장분리도적서과염색체방입0.2 mL Eppendorf관중,경거단백,Sau3A매절,병재염색체DNA편단량단가상Sau3A인공접두후,진행량륜PCR확증,득도0.4～2.0 kb지간적DNA편단.용서과적기인조표기성탐침,여단염색체확증산물진행southem잡교,표명저사서과단염색체확증편단여서과기인조DNA지간유동원성,종이증명단염색체DNA학실이피성공지확증.장제2륜PCR산물구건질립문고,득도약20 000개중조자.저시염색체미분리、미극륭기술수차재과류작물중적응용,해방법위직접종서과단조염색체DNA문고중사선분자표기전정료기출,설명이용염색체현미분리、극륭기술대서과등과류작물기인조진행연구시가행적.
The entire chromosome 1 was microdissected by using a fine glass needle under the inverted microscope, and then transferred into a collection chamber. Procedure for chromosome preparation was the squash method, in which the root-tip cells of watermelon cultivar "Zhongyuyihao" were separated and spread. The microdissected chromosome adhered to the needles was digested with proteinase K, and its DNA was extracted and digested with Sau3A. DNA ligation to a linker/primer set and PCR was carried out, in which the sequence of the linker was 5'-GATCCTGAGCTCGAATTCGACCC-3' and 5'-GGGTCGAATTCGAATTCG AGCTCAG-3]. After two rounds of PCR amplification, a smear of DNA fragments ranging from 0.4 to 2.0 kb were generated. Southern hybridization result showed that the PCR products from the single watermelon chromosome was homogeneous with the watermelon genomie DNA, indicating that DNAs from the chromosome have been successfully amplified. The second-round PCR products from chromosome of watermelon were microcloned to construct the plasmid library, including 20 000 recombinant clones. It is first reported that a simple procedure for preparation and microdissection of small chromosomes in watermelon has been successfully established. In addition, this study demonstrates that chromosome microdisseetion and cloning can be applied in genomic study of melon crops.