水稻核基因组DNA的YAC克隆和鉴定

将EcoRI部分消化的水稻(Oryza sativa L.)细胞核高分子量DNA电泳分部，回收大于200kb的片段，与内切酶消化过的酵母人工染色体(YAC)双质粒载体pJs97。pJS98DNA连接，转化酵母YPH252感受态原生质球，用ura^-,TrP^-双选择培养基直接筛选转化子，已获得2 000多个克隆。转化子DNA的southern杂交显示插入片段在200～820kb范围。

Yeast Artificial Chromosome (YAC) Cloning of HMW DNA from Rice Nuclei

A protocol to purify nuclei from etiolated seedlings of rice has been developed in order to obtain highly qualitative megabase DNA for the YAC cloning. Agarose gel blocks embeded nuclei were digested with proteinase E and the HMW DNA up to 1000kb was released. EcoR I partially digested DNA was fractionated on 1% low-point-melting agarose gel, and recovered with size larger than 200kb. The recovered DNA was ligated to dephosphorylated vectors pJS97 and pJS98 DNA, and then transformed into yeast spheroplast. Over 2 000 transformants were obtained. Colony hybridization using pUC8 and rice total DNA as probes showed that the ratio of recombinants was about 80%. The size of inserted fragments was from 200 to 820kb indicated by Southern hybridization.