粳稻染色体组基因文库的构建及DNA探针的筛选

CONSTRUCTION OF A GENOMIC LIBRARY AND SELECTION OF DNA PROBES IN JAPONICA RICE(Oryza Sativa L. )

用液氮研磨粉碎、苯酚氯仿提取纯化的方法,获得了来自粳稻品种“寒丰”的总DNA,经Pst Ⅰ限制性内切酶酶切和低熔点琼脂糖电泳分离,提取1-2kb大小的水稻DNA片段,然后在T_4连结酶的作用下,随机将其克隆到pUC_(19)质粒中,在含有X-Gal的固体培养基上,选择到515个携有水稻DNA片段的重组克隆(图1)。

The genomic library of rice cultivar 'Hanfeng' was constructed by using pUC 19 plasmid as a cloning vector. The size of inserts in these clones ranged from 0.6 to 2.7kb. Five hundred and fifteen recombinant clones from this library were randomly selected. The recombinant plasmid DNA were digested with Pst Ⅰ restriction endonuclease, and run under agarose gel. Southern blot was conducted, and the DNA were hybridized with ^(32)p dCTP labeled probes by nick translation. The probes were isolated from the total DNA of 'Hanfeng' and the chloroplast DNA (ct DNA) isolated from cotton respectively.Based on the result ofautoradiography, these clone DNA can be classified into four types: 1) chloroplast DNA, accounts for 1. 1% of total clones tested; 2) repeated sequence (9.5%); 3) multiple sequence (16%); 4) single copy DNA (73.4%). Most of these clone DNA can be used as DNA probes for rice RFLP mapping and genetic evolutional studies.