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  本文關(guān)鍵詞：黃連HPPD對其抑制劑型除草劑DTP的抗性機制分析　出處：《河北大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 黃連HPPD 除草劑抗性 定點突變 結(jié)構(gòu)預測 HPPD酶活性

【摘要】：對羥苯基丙酮酸雙加氧酶(4-Hydroxyphenylpyruvate dioxygenase,HPPD;EC.1.13.11.27)參與酪氨酸分解代謝的第二步反應,催化對羥苯基丙酮酸(HPP)形成尿黑酸(HGA),目前已開發(fā)出了多種不同類型以HPPD為靶標的抑制劑,廣泛應用于人類酪氨酸血癥的治療和雜草控制。黃連(Coptics japonica)培養(yǎng)細胞在含有HPPD抑制劑型除草劑DTP的培養(yǎng)基中生長良好,重組蛋白的體外生化分析表明這種現(xiàn)象的產(chǎn)生是由于黃連對羥苯基丙酮酸雙加氧酶(CjHPPD)對DTP具有抗性,本研究旨在對這種抗性產(chǎn)生的分子機制進行探究,為豐富抗性基因的多樣性,培育轉(zhuǎn)基因植物,提高農(nóng)作物抗性水平及作物品質(zhì)改良奠定基礎。本研究利用生物信息學方法對CjHPPD的理化性質(zhì)、信號肽、跨膜結(jié)構(gòu)域、亞細胞定位、親疏水性、二級結(jié)構(gòu)、模體及三級結(jié)構(gòu)等進行分析預測,通過分子對接技術(shù)探究DTP與CjHPPD相互作用的空間構(gòu)象。將CjHPPD蛋白質(zhì)序列與擬南芥、玉米、胡蘿卜、水麻、熒光假單胞菌、銅綠假單胞菌和AM-H4(假單胞菌屬)的HPPD序列進行比對,推測對CjHPPD除草劑抗性具有影響的氨基酸位點,對相應位點進行定點突變;將突變后的Cjhppd基因在E.coli BL21(DE3)中異源表達獲得定點突變的CjHPPD。分別測定各突變蛋白的酶活變化和對DTP的抗性變化,結(jié)合生物信息學和分子對接預測分析突變蛋白活性和抗性的分子機制。主要研究結(jié)果:結(jié)構(gòu)預測分析表明CjHPPD是由430個氨基酸殘基組成的一種無信號肽、無跨膜結(jié)構(gòu)、無卷曲螺旋、定位于微體或細胞質(zhì)的可溶性親水蛋白,α-螺旋和無規(guī)則卷曲為主要的二級結(jié)構(gòu)元件;在同源建模時,與擬南芥HPPD 1sp9晶體結(jié)構(gòu)一致性最高,達到75.41%;擬定的14個突變型H209G、A210I、L224F、T244A、I251M、P263G、M264L、Y280F、E282D、H291G、L292M、L294V、P319A和E409Q等,除了在二級結(jié)構(gòu)和親疏水性方面發(fā)生較多的變化外,信號肽、跨膜結(jié)構(gòu)域、亞細胞定位和模體等都與CjHPPD相同。HPPD酶活性測定分析顯示:定點突變的CjHPPD蛋白中,L224F、P263G、L292M的相對活性降低53%-56%;H209G、H291G相對活性降到了14.00%、20.35%,視為活性喪失;其他的9個突變相對活性增加40%-570%。對除H209G、H291G之外的突變型進行DTP抗性測定發(fā)現(xiàn),A210I、L224F、L292M和P319A突變型相對抗性降至70.33%-98.95%,其他突變型相對抗性增至112%-150%。綜合分析以上結(jié)果發(fā)現(xiàn),CjHPPD抗性的產(chǎn)生與其蛋白質(zhì)的氨基酸序列有關(guān),H209、H291是酶活性必需位點;A210、L291、L292、L294、T244、I251、Y280、E282、P263、M264、P319和E409等位點在CjHPPD對DTP的抗性表型中發(fā)揮重要作用,這些位點的改變會對蛋白質(zhì)活性口袋及其緊鄰氨基酸殘基的二級結(jié)構(gòu)、疏水性或空間位阻造成影響。
[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, 偽 -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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S482.4

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