刊期:双月刊
主管单位:中国科学院
主办单位:中国昆虫学会
                    中国科学院动物研究所

地址:北京市朝阳区北辰西路1号院5号中国科学院动物研究所
邮编:100101
电话:010-64807137
传真:010-64807137
E-Mail:entom@ioz.ac.cn
刊号:ISSN 2095-1353
        CN 11-6020/Q
国内发行代号:2-151
国际发行代号:BM-407
发行范围:国内外公开发布
定价:100元/册
定价:600元/年
银行汇款:中国工商银行北京海淀西区支行
户名:中国科学院动物研究所
帐号:0200 0045 0908 8125 063

您所在位置:首页->过刊浏览->2016年53卷第4期


苜蓿夜蛾葡萄糖氧化酶cDNA的克隆、表达及特性研究
cDNA cloning, expression and characterization ofglucose oxidase from Heliothis viriplaca
雷 蕾** 赵奎军 高艳玲 李莉莉 樊 东***
点击:520次 下载:64次
作者单位:东北农业大学农学院,哈尔滨 150030
中文关键词:苜蓿夜蛾,葡萄糖氧化酶,克隆,表达,酶活性
英文关键词: Heliothis viriplaca, glucose oxidase, cloning, expression, enzyme activity
中文摘要:

【目的】 从苜蓿夜蛾Heliothis viriplaca体内克隆葡萄糖氧化酶(Glucose oxidase, GOX)基因cDNA序列,并进行原核表达及活性测定。同时对GOX在不同组织的特异表达及对葡萄糖诱导反应的特性进行研究。【方法】 以苜蓿夜蛾为材料提取总RNA,利用RT-PCRcDNA末端快速扩增技术,扩增苜蓿夜蛾GOX基因全长cDNA序列。利用原核表达载体pET-21b在大肠杆菌中表达苜蓿夜蛾的GOX基因,并用Ni-NTA亲和层析柱将带His-tag的目的蛋白进行纯化,再利用梯度透析法进行复性,以葡萄糖为底物进行酶的活性测定。同时利用Real-time PCR分析GOX的组织特异性表达和对葡萄糖的诱导反应。【结果】 cDNA序列有2 154个碱基,开放阅读框1 824个碱基,编码607个氨基酸组成的多肽,分子量为67.04 ku,多肽的等电点为5.13。该序列命名为HvGOX,在GenBank的登录号为KT907054。序列分析表明,HvGOX的氨基酸序列与其他昆虫的GOX氨基酸序列高度同源。该基因在大肠杆菌表达系统中成功地进行了诱导表达,表达出与预测的蛋白分子量相符的融合蛋白。由原核表达载体表达后的蛋白经过变性、纯化和复性后有活性。Real-time PCR 结果表明,该基因在苜蓿夜蛾的不同组织均有mRNA水平的特异表达,其中在唾腺中的表达量最高;同时用0.01%0.1%1%10%葡萄糖溶液浸泡的大豆叶喂食的幼虫,幼虫体内的GOX的表达均被诱导,且在10%的浓度时诱导表达量最高。【结论】 本研究在苜蓿夜蛾体内获得一个新的葡萄糖氧化酶基因,该结果为进一步研究葡萄糖氧化酶在昆虫体内的生物功能奠定基础。

英文摘要:

[Objectives]  To obtain the full-length cDNA sequence of glucose oxidase (GOX) from Heliothis viriplacaand determine the GOX activity in the prokaryotic expression system, mRNA expression levels in different tissues and the characterization of glucose induction. [Methods]  Total RNA was isolated from the larva of H. viriplaca. The RT-PCR and rapid amplification of cDNA ends techniques were used to clone the full-length GOX cDNA sequence. The E. coli prokaryotic expression system was used to express the cDNA sequence. The recombinant protein, with a His-tag, was purified by Ni-NAT affinity chromatography. After purification, the recombinant protein was renatured using the gradient dialysis technique. The activity of GOX protein was determined with glucose as the substrate. Real-time PCR was used to analyze mRNA expression levels in different tissues and the reaction of glucose induction. [Results]  The cDNA sequence of GOX was 2 154 bp, including an open reading frame of 1 824 bp encoding a polypeptide of 607 amino acids with an estimated molecular mass of 67.04 ku and an estimated isoelectric point of 5.13. The cDNA sequence has been deposited in GeneBank (accession No. KT907054) and designated as HvGOX. SDS-PAGE revealed that HvGOX was successfully expressed in E. coli. The expressed protein was active after denaturation, purification and renaturation. Sequence analysis indicates that HvGOX shares extensive similarities with that of other insects. Real-time PCR results showed that the mRNA transcripts were expressed in different tissues, with the highest expression in the salivary gland. GOX expression in larvae was induced by feeding them soybean leaves soaked in 0.01%, 0.1%, 1% and 10% glucose solution; expression was highest when larvae were fed leaves soaked in 10% glucose solution. [Conclusion]  A novel GOX cDNA sequence was obtained from H. viriplaca. The results provide a foundation for further research on the biological function of GOX in insects.

读者评论

      读者ID: 密码:   
我要评论:
版权所有©2018应用昆虫学报》编辑部 京ICP备:10006425号
本系统由北京菲斯特诺科技有限公司设计开发
您是本站第3272976名访问者