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以柑橘属Swingle分类系统界定的16种柑橘物种的基因组DNA为材料,构建了研制柑橘DArT芯片的DNA探针文库,进而从中随机挑选出15 000条探针用于柑橘DArT芯片的开发.柑橘物种的基因组DNA采用改良CTAB法提取并等量混合,经Eco RI和Mse I双酶切后连接接头序列,再使用选择性引物进行PCR扩增,并将扩增产物转化大肠杆菌,构建探针文库.从探针文库中随机挑选出15 000个阳性单克隆,通过PCR扩增和纯化获得了15 000条用于制备DArT芯片的DNA探针.凝胶电泳检测显示所制备的DNA探针的纯度和浓度均满足了研制DArT芯片的要求.为采用DArT芯片快速鉴定柑橘物种并研究其进化关系奠定了基础.
Based on the genomic DNA of 16 citrus species defined by the Swingle classification system of citrus, DNA probe library of citrus DArT chip was constructed and 15 000 probes were randomly selected for the development of citrus DArT chip. The genomic DNA was extracted by the modified CTAB method and mixed equally. After digested with EcoRI and Mse I, the adapter sequence was ligated. Then the selective primers were used for PCR amplification. The amplified product was transformed into E. coli to construct a probe library 15 000 positive clones were randomly selected from the probe library and 15 000 DNA probes for preparing DArT chips were obtained by PCR amplification and purification.The gel electrophoresis test showed that the purity of the prepared DNA probe And concentration all meet the requirement of developing DArT chip, which lays the foundation for the rapid identification of citrus species by DArT chip and the study of its evolutionary relationship.