| 禾谷镰刀菌14α-脱甲基酶的原核表达与纯化 |
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| 引用本文： | 钱恒伟,黄金光.禾谷镰刀菌14α-脱甲基酶的原核表达与纯化[J].青岛农业大学学报(自然科学版),2017(3):169-176. |
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| 作者姓名： | 钱恒伟 黄金光 |
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| 作者单位： | 1. 青岛农业大学农学与植物保护学院,山东 青岛,266109;2. 青岛农业大学农学与植物保护学院,山东 青岛 266109;山东省植物病虫害综合防控重点实验室,山东 青岛 266109 |
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| 基金项目： | 国家自然基金(31471735)，国家重点研发计划重点专项黄淮海冬小麦化肥农药减施技术集成研究与示范(2017YFD0201705)，青岛市现代农业产业技术体系(6622316106) |
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| 摘 要： | 近年来,三唑类杀菌剂成为生产上防治小麦赤霉病(Fusarium Head Blight,FHB)的主要药剂,主要作用于甾醇生物合成中的14α-脱甲基酶(CYP51),使真菌麦角甾醇的合成受阻,从而破坏细胞膜功能,起到杀菌的功能.本研究的目的是采用结构生物学的方法探究禾谷镰刀菌(Fusarium graminearum)中14α-脱甲基酶(FgCYP51B)的三维结构、生化功能以及其与三唑类杀菌剂的互作机制,进一步阐释禾谷镰刀菌对三唑类杀菌剂潜在的抗性风险.以禾谷镰刀菌(F.graminearum)的cDNA为模板,根据14α-脱甲基酶基因(FgCYP51B)序列设计特异性引物进行PCR扩增,克隆了FgCYP51B基因并构建了3个全长表达载体pHAT2-CYP51B、pETM-20-CYP51B和pETM-30-CYP51B以及3个蛋白截短体表达载体pHAT2-CYP51B50-527,pETM-20-CYP51B50-527和pETM-30-CYP51B50-527,将重组表达载体转化到大肠杆菌BL21(DE3)和Rosetta(DE3),并利用异丙基硫代半乳糖苷(IPTG)进行诱导表达.结果表明:全长FgCYP51B蛋白在BL21(DE3)和Rosetta(DE3)中均不表达;重组蛋白pHAT2-CYP51B50-527在大肠杆菌中不表达,而重组蛋白pETM-20-CYP51B50-527和pETM-30-CYP51B50-527成功表达,并通过亲和层析、离子交换与分子筛对重组蛋白进行纯化.该蛋白的表达与纯化为其结构功能的研究奠定了基础.
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| 关 键 词： | 禾谷镰刀菌 FgCYP51B 原核表达 蛋白纯化 亲和层析 离子交换 分子筛 |
Prokaryotic Expression and Purification of Sterol 14a-demethylase from Fusarium graminearum |
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| QIAN Hengwei,HUANG Jinguang.Prokaryotic Expression and Purification of Sterol 14a-demethylase from Fusarium graminearum[J].Journal of Laiyang Agricultural College,2017(3):169-176. |
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| Authors: | QIAN Hengwei HUANG Jinguang |
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| Abstract: | In recent years, azole fungicides have beenused tocontrol of Fusarium Head Blight.Azolesinhibit the biosynthesis of ergosterol in fungi,which is essential for cell membrane fluidity and permeability, forthe key sterol 14a-demethylase (CYP51) is the target of azole fungicides.The aim of this researchwas to investigate the interaction between the CYP51 protein of F.graminearum and azoles, and to explore the possible risk and mechanism of resistance.Special primers were designed based on the sequences of codon ofFgCYP51B.The gene of FgCYP51B was obtained by PCR amplificationwith the template of F.graminearum cDNA,and then three full-length expression vectors pHAT2-CYP51B, pETM-20-CYP51B, pETM-30-CYP51B and three truncated protein expression vector pHAT2-CYP51B50-527, pETM-20-CYP51B50-527, pETM-30-CYP51B50-527 were constructed respectively.Recombinant expression vectors were transformed into Escherichia coliBL21(DE3) and Rosetta(DE3) and induced by IPTG.The SDS-PAGE detection showed that the full-length FgCYP51B protein was not expressed in E.coli;The recombinant protein pHAT2-CYP51B50-527 was not expressed, but the recombinant protein pETM-20-CYP51B50-527and pETM-30-CYP51B50-527 were expressed, and the recombinant protein was purified by affinity chromatography, ion exchange and gel filtration.The expression and purification of the protein established an important platform and basis for further study of the protein function. |
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| Keywords: | Fusarium graminearum FgCYP51B prokaryotic expression protein purification affinity chromatography ion exchange gel filtration |
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