胡萝卜抗冻蛋白基因的克隆及其在E.coli中的表达

对中国的一个胡萝卜品种进行了冷诱导前后幼苗总蛋白组成的比较、分析 ,SDS聚丙烯酰胺凝胶电泳的结果表明两者在相对分子质量 36 0 0 0附近有一条蛋白带的差别。由此推测实验的胡萝卜品种在冷诱导过程中有抗冻蛋白 (AFP)表达。根据相关的研究结果设计引物 ,以胡萝卜幼苗基因组DNA为模板 ,通过PCR扩增得到了长 10 99bp的抗冻蛋白基因。测序结果表明其与GenBank公布的afp序列仅有 3个碱基的差别。将此抗冻蛋白基因克隆进大肠杆菌表达载体pGEX4T1,在IPTG的诱导下表达出了含有AFP的约 6 0 0 0 0的融合蛋白 ,证明该AFP基因在原核表达系统中可以正常表达

Cloning and Expression of an Antifreeze Protein Gene in Carrot

Composition of total proteins in cold_induced carrot seedlings was compared with that in non_induced carrot seedlings by SDS PAGE. A different protein band, about 36 000 appeared. This result indicated that antifreeze proteins (AFPs) were expressed in cold_induced carrot seedlings. Based on what was reported by Dawn Worrall and Knut Meyer, the gene encoding this antifreeze protein which is 1 099 bp long, was amplified by PCR using genomic DNA of carrot seedlings as template. Sequencing results show that there is only a difference of three bases between this cloned gene and that published on GenBank. This antifreeze protein gene (AFP gene) was cloned in the expression vector (pGEX4T1) of E coli. A fusion protein, about 60 000, could be detected after IPTG_induction.