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从不亲和野生花生ArachisglabrataBenth中提取DNA,在多重序列比对的基础上设计PCR引物,成功扩增出约350、750bp的两个片段,与DES和RS基因片段预期分子量相符。PCR产物与T载体连接,转化受体菌XL1 Blue,获得了白色菌落。挑取白色菌落在液体培养基中过夜培养,经TTE溶液和热处理后进行PCR扩增,获得了载有目的基因片段的阳性重组子。测序和序列比对分析的结果说明,DES和RS基因片段克隆成功。这是首次从不亲和野生花生A.glabrataBenth中克隆出的基因片段。该片段可用作探针克隆相关基因全编码区。
DNA was extracted from incompatible wild peanut ArachisglabrataBenth. PCR primers were designed based on multiple sequence alignments. Two fragments of about 350,750bp were successfully amplified, which were consistent with expected molecular weight of DES and RS gene fragments. The PCR product was ligated with T vector and transformed into recipient strain XL1 Blue to obtain white colonies. The white colonies were picked for overnight culture in liquid medium. After TTE solution and heat treatment, PCR amplification was carried out to obtain the positive recombinant containing the gene fragment of interest. The results of sequencing and sequence alignment analysis showed that the cloning of DES and RS gene fragments was successful. This is the first gene cloned from A. glabrataBenth, a non-compatible wild peanut. This fragment can be used as a full-length coding region for probe cloning genes.