用聚合酶链式反应扩增抗冻肽基因并在双元表达载体中克隆

Amplification of an Antifreeze Peptide Gene by PCR and Its Cloning in the Binary Expression Vectors Erden Dalai

抗冻肽基因导入植物的第一步合成了５＇－末端分别带有限制性酶ｘｂａＩ和ＫｐｎＩ识别位点的一对引物，以美洲拟鲽抗冻肽的ｃＤＮＡ克匪为模板，用锚定聚合酶链式反应扩增了抗冻肽基因，并克隆到细菌表达载体ｐＧＥＭ３２ｆ（—）和植物表达盒式载体ｐＧＡ６４３中，以限制性酶切分析和菌落杂交技术证明了转化子质粒ＤＮＡ中的含有抗冻肽基因，又用ＰＣＲ方法扩增了转化子的抗冻肽基因，从而证实了克隆成功．

n order to intrduce a gene for antifreeze peptide(AFP)under appropriate regulary controlinto the plant tissues,a XbaI/KpnI fragment containing AFP-coding gene is amplified by PCRfrom pCT5,a cDNA clone of winter flounder antifreeze peptide in PBR322,and subcloned intobacterial expression vector pGEM-3Zf( )and plant expression cassette of pGA643.These sub-clones are confirmed by the restriction enzyme digestion analysis and the colony hybridizationtechnique.