洛氏脊漠甲抗冻蛋白基因的克隆、表达和功能检测  

Cloning and activity analysis of insect antifreeze protein gene from Pterocoma loczyi (Coleoptera:Tenebrionidae)

作　　者：马纪[1] 王芸[1] 刘忠渊[1] 张富春[1] 

机构地区：[1]新疆大学生命科学与技术学院,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》2008年第5期480-485,共6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(XJDX0201-2008-04)

摘　　要：为了研究抗冻蛋白基因是否在新疆荒漠昆虫洛氏脊漠甲Pterocomaloczyi中存在,利用RT-PCR技术克隆获得了洛氏脊漠甲抗冻蛋白的cDNA片段,命名为Plafp743。测序结果表明洛氏脊漠甲Plafp743所编码的蛋白质由94个氨基酸组成,蛋白序列呈现规则结构CTX1X2X3X4CX5X6X7X8X9。利用原核表达载体构建重组质粒pGEX-4T-1-Plafp743,转化Escherichia coliBL21进行融合蛋白表达,SDS-PAGE结果表明抗冻蛋白PLAFP基因以可溶性融合蛋白形式表达,相对分子质量36kD。用Glutathione Sepharose4B亲和柱对表达蛋白进行纯化后,通过赤翅甲Dendroidescanadensis抗冻蛋白的小鼠抗血清进行Western blot分析,结果表明纯化的GST-PLAFP融合蛋白与赤翅甲抗血清能够发生特异性的免疫反应。大肠杆菌的低温抗冻保护实验结果证明,30μg/mL的GST-PLAFP在-6℃对细菌具有显著的保护作用,且随着抗冻蛋白浓度的增加,抗冻保护作用的效果也随之增加。本研究首次报道了从荒漠昆虫洛氏脊漠甲扩增得到抗冻蛋白基因,提示抗冻蛋白可能是荒漠昆虫普遍采取的过冬生存策略,为进一步开发利用昆虫抗冻蛋白奠定了基础。To investigate antifreeze protein gene in the beetles living in Xinjiang Junggar desert, Pterocoma loczyi, a large-size desert beetle, was chosen for antifreeze gene cloning. RT-PCR technique was employed to obtain Pterocoma loczyi antifreeze protein gene. One of the obtained cDNA fragment was named Plafp743. zSequence analysis showed that Plafp743 encodes 112 amino acids with the repeated constructive sequence CTX1X2X3X4CX5 X6X7 X8 X9, which is the special motif for some of the known insects antifreeze proteins. Recombinant plasmid pGEX-4T-1-Plafp743 was transferred into Escherichia coli BL21 and induced by IPTG. The expressive product was analyzed by SDS-PAGE, and the result showed that pGEX-4T-1-Plafp-743 was expressed in fusion protein with relative molecular mass of 36 kD. The fusion protein, named GST-PLAFP, was purified by Glutathione Sepharose 4B, and then identified by Western blotting with mouse antiserum against antifreeze protein from Dendroides canadensis. Then, GST-PLAFP with different concentrations was added to E. coli culture for various days at - 6 ℃ to check its cryoprotective effect on bacteria survival at subzero temperatures. This cryoprotective experiment showed that the fusion protein GST-PLAFP could protect bacteria from freezing to death at concentrations as low as 30 μg/mL. The results suggest that AFP might be a widely adopted strategy for overwintering of desert beetles.

关 键 词：洛氏脊漠甲 抗冻蛋白 克隆 序列分析 原核表达 抗冻保护 

分 类 号：Q966[生物学—昆虫学]