新疆准噶尔小胸鳖甲抗冻蛋白基因的克隆和抗冻活性分析  

Cloning of a cDNA encoding antifreeze protein in Microdera punctipenis dzunarica (Coleoptera: Tenebrionidae) and its activity assay

作　　者：赵干[1] 马纪[1] 薛娜[1] 杨长庚[1] 专芳芳[1] 张富春[1] 

机构地区：[1]新疆大学生命科学与技术学院分子生物学重点实验室,新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《昆虫学报》2005年第5期667-673,共7页Acta Entomologica Sinica

基　　金：科技部重大基础研究前期研究专项(2003CCA01000);新疆生物资源基因工程重点实验室开放课题(XJDX0201-200403)

摘　　要：根据GenBank中已发表的昆虫抗冻蛋白基因的保守序列设计引物,利用RT-PCR技术结合3’-RACE扩增的方法,从新疆荒漠昆虫准噶尔小胸鳖甲Microderapunctipenisdzunarica中获得了长约294bp不含信号肽的抗冻蛋白cDNA片段,命名为MpAFP5,其全长序列为363bp(GenBank注册号为AY821792)。基因测序结果表明,MpAFP5cDNA片段与加拿大拟步甲DendroidescanadensisAFP8基因片段、黄粉甲TenebriomolitorTHP4-9基因片段的核苷酸同源性分别达68.4%和71.8%,氨基酸序列同源性分别达70%和81%。将MpAFP5构建到原核表达载体pGEX4T-1中,重组质粒pGEX4T-1-MpAFP5在大肠杆菌BL21(DE3)中能够表达融合抗冻蛋白,SDS-PAGE分析显示融合蛋白的分子量约为37kD,Western印迹分析证明MpAFP5在大肠杆菌中正确表达。细菌的抗冻实验结果显示准噶尔小胸鳖甲融合抗冻蛋白对细菌具有显著的抗冻保护作用,保护效果与抗冻蛋白剂量呈正相关性。For cloning antifreeze protein （AFP） gene from a desert darkling beetle Microdera punctipenis dzunarica in Xinjiang, the primers were designed according to the core sequence of AFP gene deposited in GenBank, and the cDNA fragment about 294 bp named as MpAFP5 was amplified with the RT-PCR and 3＇-RACE technique. Sequence analysis revealed that the cloned cDNA fragment named as MpAFP5 coded the mature peptide of AFP. The full sequence of MpAFP5 （AY821792） was about 363 bp. Compared with the Dendroides canadensis AFP-8 and Tenebrio molitor thermal hysteresis protein （THP） isoform 4-9 genes, it had 68.4% and 71.8% identity in gene level, and 70% and 81% identity in protein level respectively. The MpAFP5 gene was then cloned into prokaryotic plasmid pGEX4T-1 and expressed in E. coli BL21 （DE3）. SDS-PAGE analysis indicated that the fusion antifreeze protein was expressed in E. coli with a molecular weight of about 37 kD. Western-blot analysis showed that MpAFP5 was expressed correctly. Tests on low temperature protection to bacteria showed that the insect antifreeze protein could protect bacteria from the low temperature damages, and the protective effect was correlated with the antifreeze protein concentration.

关 键 词：准噶尔小胸鳖甲 抗冻蛋白 RT-PCR 融合蛋白 抗冻活性 

分 类 号：Q966[生物学—昆虫学]