黄粉甲抗冻蛋白基因afp72 cDNA的克隆和序列分析.pdfVIP

安徽农业科学，Journal ofAnhui Agri．Sci．2008，36(14)：5763-5766 责任编辑 郑丹丹 责任校对 马君叶 黄粉甲抗冻蛋白基因aD72 eDNA的克隆和序列分析 闫清华·，-，邵强卜，李延兰2，丰慧根 (1．河南师范大学生命科学学院，河南新乡453007；2．新乡医学院生命科学技术系，河南新乡453003) 摘要 [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4P72,-)~-构建克隆重组质粒pucm 诉72。经双酶切后，将其与表达栽体pMAL-p2X连接并转化到大肠杆菌TBI，构 建表达重组质粒pMAL_p2x诉72。[结果] 72cDNA长度为216 bp；afp72的测序序列与GenBank中编码84个氨基酸的黄粉甲 ps 序列的同源性为89．48％； )72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， 4P72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。 关键词 黄粉甲；抗冻蛋白； 72；3Lg~；表达栽体 、 中图分类号 Q785 文献标识码 A 文章编号 o517—6611(2008)14—05763—04 Cloning of Antifreeze Protein Gene afp72 cDNA in Tenebrio molitor and Its Expression Vector Construction YAN Qing hua et al(College．of Lifo Science，Henan Normal University，Xinxiang,Henan 453007) Abstract [Objective]The research aimed to lav the foundation for studying the expression in Vitro and biological functions of antifreeze protein gene斫)72． e山od]Total RNA was isolated from fat bodv of Tenebrio molitor and antifreeze protein gene afp72 was ci【】ned by RT,PCR．The recombinant cloning vector pUCm-T-alp72 was constructed．After double-enzyme digestion，it was linked with expression vector pMALp2X and transformed into Escherichia coli TBI to construct recombinant expressionvector pMAL-p2X-a~72．[Result]The 1ength of afp72cDNA was 216 bp．The homology between me tested 72sequence and the ps sequence of molitor tl1at encoded 84 amino acids on GenBank wet，site was 89．48％． )72 gene was probaly、from the mutation or deletion of other antifreeze protein gene in molhor．It was proved that AFP72 was an antifreeze protein and q 72 was an antifreeze protein gene．The construction of recombinant expression vector pMAL-p2X-#p72 was successfu1． 『Conclusion]This research provided materials f0r constructing the transgenic plant with antifreeze protein by using gene engineering method． K