黄连HPPD对其抑制剂型除草剂DTP的抗性机制分析

发布时间：2018-01-16 06:04

本文关键词：黄连HPPD对其抑制剂型除草剂DTP的抗性机制分析　出处：《河北大学》2017年硕士论文　论文类型：学位论文

【摘要】：对羟苯基丙酮酸双加氧酶(4-Hydroxyphenylpyruvate dioxygenase,HPPD;EC.1.13.11.27)参与酪氨酸分解代谢的第二步反应,催化对羟苯基丙酮酸(HPP)形成尿黑酸(HGA),目前已开发出了多种不同类型以HPPD为靶标的抑制剂,广泛应用于人类酪氨酸血症的治疗和杂草控制。黄连(Coptics japonica)培养细胞在含有HPPD抑制剂型除草剂DTP的培养基中生长良好,重组蛋白的体外生化分析表明这种现象的产生是由于黄连对羟苯基丙酮酸双加氧酶(CjHPPD)对DTP具有抗性,本研究旨在对这种抗性产生的分子机制进行探究,为丰富抗性基因的多样性,培育转基因植物,提高农作物抗性水平及作物品质改良奠定基础。本研究利用生物信息学方法对CjHPPD的理化性质、信号肽、跨膜结构域、亚细胞定位、亲疏水性、二级结构、模体及三级结构等进行分析预测,通过分子对接技术探究DTP与CjHPPD相互作用的空间构象。将CjHPPD蛋白质序列与拟南芥、玉米、胡萝卜、水麻、荧光假单胞菌、铜绿假单胞菌和AM-H4(假单胞菌属)的HPPD序列进行比对,推测对CjHPPD除草剂抗性具有影响的氨基酸位点,对相应位点进行定点突变;将突变后的Cjhppd基因在E.coli BL21(DE3)中异源表达获得定点突变的CjHPPD。分别测定各突变蛋白的酶活变化和对DTP的抗性变化,结合生物信息学和分子对接预测分析突变蛋白活性和抗性的分子机制。主要研究结果:结构预测分析表明CjHPPD是由430个氨基酸残基组成的一种无信号肽、无跨膜结构、无卷曲螺旋、定位于微体或细胞质的可溶性亲水蛋白,α-螺旋和无规则卷曲为主要的二级结构元件;在同源建模时,与拟南芥HPPD 1sp9晶体结构一致性最高,达到75.41%;拟定的14个突变型H209G、A210I、L224F、T244A、I251M、P263G、M264L、Y280F、E282D、H291G、L292M、L294V、P319A和E409Q等,除了在二级结构和亲疏水性方面发生较多的变化外,信号肽、跨膜结构域、亚细胞定位和模体等都与CjHPPD相同。HPPD酶活性测定分析显示:定点突变的CjHPPD蛋白中,L224F、P263G、L292M的相对活性降低53%-56%;H209G、H291G相对活性降到了14.00%、20.35%,视为活性丧失;其他的9个突变相对活性增加40%-570%。对除H209G、H291G之外的突变型进行DTP抗性测定发现,A210I、L224F、L292M和P319A突变型相对抗性降至70.33%-98.95%,其他突变型相对抗性增至112%-150%。综合分析以上结果发现,CjHPPD抗性的产生与其蛋白质的氨基酸序列有关,H209、H291是酶活性必需位点;A210、L291、L292、L294、T244、I251、Y280、E282、P263、M264、P319和E409等位点在CjHPPD对DTP的抗性表型中发挥重要作用,这些位点的改变会对蛋白质活性口袋及其紧邻氨基酸残基的二级结构、疏水性或空间位阻造成影响。
[Abstract]:4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenylpyruvate dioxygenase); EC.1.13.11.27) is involved in the second step of tyrosine catabolism and catalyzes the formation of tetrahydropyruvate (HPP) to form melanoic acid (HGA). At present, many different types of inhibitors targeting HPPD have been developed. Widely used in the treatment of human tyrosinemia and weed control. Coptics japonica. The cultured cells grew well in the medium containing HPPD inhibitor type herbicide DTP. Biochemical analysis of recombinant protein in vitro showed that this phenomenon was due to the resistance of Coptis chinensis to DTP. The purpose of this study is to explore the molecular mechanism of this resistance in order to enrich the diversity of resistance genes and to cultivate transgenic plants. In this study, the physicochemical properties, signal peptide, transmembrane domain, subcellular location and hydrophobicity of CjHPPD were studied by bioinformatics. The secondary structure, motifs and tertiary structures were analyzed and predicted, and the spatial conformation of the interaction between DTP and CjHPPD was studied by molecular docking technique. The sequence of CjHPPD protein was combined with Arabidopsis thaliana and maize. The HPPD sequences of carrot, Ramie, Pseudomonas fluorescens, Pseudomonas aeruginosa and Pseudomonas aeruginosa (Pseudomonas aeruginosa) were compared, and the amino acid sites affecting CjHPPD herbicide resistance were deduced. The site-directed mutation of the corresponding loci was carried out. Mutation of Cjhppd gene in E. coli BL21DE3. CjHPPDs with site-directed mutagenesis were obtained. The enzyme activity and the resistance to DTP of the mutant proteins were determined. Molecular mechanisms for predicting the activity and resistance of mutant proteins by bioinformatics and molecular docking. Main results:. Structural prediction analysis showed that CjHPPD was a signal free peptide composed of 430 amino acid residues. No transmembrane structure, no curl helix, soluble hydrophilic protein located in microbody or cytoplasm, 伪 -helix and irregular crimp are the main secondary structural elements. In homologous modeling, the crystal structure of Arabidopsis thaliana HPPD 1sp9 was consistent with that of Arabidopsis thaliana, and reached 75.41. Fourteen mutants, H209G, A210IHN, L224FN, T244Afen, I251MN, P263GnM264L, Y280FU, E282D, H291G, have been developed. In addition to the changes in secondary structure and hydrophobicity, signal peptides and transmembrane domains were observed in L292MU, L294V, P319A and E409Q. The activity analysis of subcellular localization and motif was the same as that of CjHPPD. The results showed that L224FU P263G was in the CjHPPD protein of site-directed mutation. The relative activity of L292M was decreased by 53-56; The relative activity of H209GN H291G was reduced to 14.00 and 20.35g, which was regarded as loss of activity. The relative activity of the other 9 mutants increased by 40- 570.The DTP resistance test of the mutants other than H209G / H291G revealed that A210IXL224F was detected. The relative resistance of L292M and P319A mutant decreased to 70.33-98.95, and the other mutants increased to 112-150. The production of CjHPPD resistance is related to the amino acid sequence of its protein. H209 H291 is an essential site for enzyme activity. A210,L291,L292,L294,T244,I251,Y280,E282,P263,M264. Sites such as P319 and E409 play an important role in the phenotype of CjHPPD resistance to DTP. The changes of these sites will affect the secondary structure of protein active pocket and its adjacent amino acid residues. Hydrophobicity or steric hindrance.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S482.4

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