小冰麦异附加系中携带BYDV抗性基因染色体的微分离及其文库的建立

Microdissection and Microcloning of the Addition Chromosome in Wheat-wheatgrass Alien Addition Line TAI-27 Containing BYDV Resistant Gene

采用微玻璃针法显微分离了小冰麦异附加系TAI 2 7中附加有中间偃麦草 (天蓝冰草 )的一对染色体 ,这对染色体上携带有抗大麦黄矮病的抗性基因。经过蛋白酶K消化和两轮寡聚核苷酸引物 PCR (degenerateoligonucleotideprimedPCR ,DOP PCR)扩增后 ,用TAI 2 7和中间偃麦草的总DNA为探针的Southern杂交证明PCR产物确是来自这对小染色体。第二轮PCR产物被连接至pGEM Tvector中并转化大肠杆菌 ,获得小染色体DNA文库。初步分析文库共有 2× 10 5个克隆子 ,插入片段长度在 2 50～ 12 0 0bp之间 ,平均 530bp ,其中单、低拷贝序列占 57% ,中、高拷贝序列占 4 3%。

WT5BZ]The wheat wheatgrass alien addition line TAI 27 contains a pair of chromosomes derived from Thinopyrum intermedium. These pair of chromosomes were microdissected by grassneedle and digested with proteinase K. The DNA from the alien chromosome were amplified by degenerated oligonucleotide primed PCR (DOP PCR) for two rounds. With Thinopyrum intermedium and TAI 27 genomic DNA as probes the results of Southern blotting showsl that the products of the second round PCR were derived from the alien chromosome of TAI 27. The second PCR products were ligated to pGEM T vector and the chromosome specific DNA library had been got. This library estimated to have 2×10 5 clones. The size of clone inserts varied from 250 to 1200bp, with an average of 530bp. The results of dot blotting showed that the library contains 57% single/low copy sequence clones and 43% medium/high copy sequence clones.