黄粉甲抗冻蛋白基因afp72 cDNA的克隆和序列分析

目的]为抗冻蛋白基因afp72的体外表达和生物学功能研究奠定基础。方法]从黄粉甲脂肪体中提取总RNA,通过RT-PCR克隆抗冻蛋白基因afp72,并构建克隆重组质粒pUCm-T-afp72。经双酶切后,将其与表达载体pMAL-p2X连接并转化到大肠杆菌TBI,构建表达重组质粒pMAL-p2X-afp72。结果]afp72 cDNA长度为216 bpa;fp72的测序序列与GenBank中编码84个氨基酸的黄粉甲afps序列的同源性为89.48%a;fp72基因可能来自其他黄粉甲抗冻蛋白基因的突变或缺失;从蛋白水平上证实AFP72是一个抗冻蛋白,afp72是一个抗冻蛋白基因;表达重组质粒pMAL-p2X-afp72的构建是成功的。结论]该研究为利用基因工程方法构建抗冻蛋白转基因植物提供了材料。

Cloning of Antifreeze Protein Gene afp72 eDNA in Tenebrio molitor and Its Expression Vector Construction

Objective] The research aimed to lay the foundation for studying the expression in vitro and biological functions of antifreeze protein gene afp72.Method] Total RNA was isolated from fat body of Tenebrio molitor and antifreeze protein gene afp72 was cloned by RT-PCR.The recombinant cloning vector pUCm-T-afp72 was constructed.After double-enzyme digestion,it was linked with expression vector pMAL-p2X and transformed into Escherichia coli TBI to construct recombinant expressionvector pMAL-p2X-afp72.Result] The length of afp72cDNA was 216 bp.The homology between the tested afp72sequence and the afps sequence of T.molitor that encoded 84 amino acids on GenBank website was 89.48%.afp72 gene was probaly from the mutation or deletion of other antifreeze protein gene in T.molitor.It was proved that AFP72 was an antifreeze protein and afp72 was an antifreeze protein gene.The construction of recombinant expression vector pMAL-p2X-afp72 was successful.Conclusion] This research provided materials for constructing the transgenic plant with antifreeze protein by using gene engineering method.