【作者】 赵莉；

【导师】 刘德立；

【作者基本信息】 华中师范大学 ， 农药学， 2007， 博士

【摘要】 柑桔青霉病（Penicillium italicum Wehmer）和绿霉病（Penicillium digitatum Sacc）是柑桔果实在贮运期发生最严重的病害。目前农业上广泛使用的甾醇生物合成抑制剂（sterolbiosynthesis inhibitors,SBIs）类杀菌剂主要为14α-脱甲基酶抑制剂（14α-demethylation inhibitors,DMIs）。羊毛甾醇14α-去甲基化酶(CYP51,P450_14DM)是唯一广泛存在于细菌、真菌、植物和动物中的细胞色素P450家族成员,它被视为最古老的细胞色素P450家族成员,是氮唑类抗真菌药物作用靶标酶。CYP51是真菌细胞膜麦角甾醇生物合成过程中的一个关键酶,它催化甾醇的14α-去甲基化反应。抑制真菌甾醇14α-去甲基化酶将会阻碍真菌麦角甾醇的合成,而麦角甾醇是真菌细胞膜基本成份,它的缺乏将导致膜结构的破坏和功能消失,最终导致真菌死亡。目前农业上DMIs类杀菌剂即是通过抑制真菌甾醇14α-去甲基化酶而发挥杀菌抑菌的作用。本研究以我国南方重要经济作物柑桔的病原菌绿霉菌（P.digitatum Sacc）为实验材料,采用基因工程技术克隆了柑桔绿霉菌CYP51基因,进行了高效表达和功能研究。同时,建立了柑桔绿霉菌CYP51蛋白的三维空间结构模型,并利用结合光谱法对有机小分子化合物的活性进行了筛选。本文主要得到以下结果:1.从柑桔绿霉菌（P.digitatum Sacc）中克隆了氮唑类杀真菌剂的靶标酶CYP51（PdCYP51）。该基因与文献中报道的柑桔绿霉菌CYP51基因（No.AB030178）相比有四个核苷酸不同,分别为C1370→T,A1604→G,G1648→A和G1768→T,该基因序列已提交Genbank（登记号为DQ355161）。这四个不同的核苷酸相应地导致四个氨基酸的不同,分别是P345→L,E423→G,G438→R和V478→L。该基因编码的蛋白质由516个氨基酸组成,同源比对结果表明,该蛋白与柑桔绿霉菌PD5（BAB03658,Pd2）同源性为99%,与意大利青霉P450-14DM（Q12664,Pi）蛋白同源性为86%,与烟曲霉Aspergillus fumigatus（AAF32372,Af）和费希新萨托菌Neosartorya fischeri（XP₀01267338,Nf）同源性为67%,含有CYP51家族蛋白的特征保守域SRS1（YxxF/LxxPxFGxxVxF/YD/a）和SRS4（GO/nht/sS）及P450家族保守域HBR（FxxGxxxCxG）。序列分析表明四个突变氨基酸位于PdCYP51的保守结构域之外,生测结果表明这四个突变氨基酸与该菌的抗性相关性不大。2.以结核分枝杆菌CYP51（Mycobacterium tuberculosis,MTCYP51）晶体结构为模板,利用SYBYL7.0软件包中的FUGUE模块成功地构建了柑桔绿霉菌CYP51蛋白的三维空间结构模型,PdCYP51蛋白三维空间模型与MTCYP51蛋白晶体结合叠合结果显示PdCYP51血红素辅基附近氨基酸具有很好重叠性。CYP51是一类跨膜蛋白,在真菌的麦角甾醇生物合成过程中起着非常重要的作的用。对其跨膜区分析表明,PdCYP51N端66个氨基酸跨膜两次。构建了重组表达质粒pET-PdCYP51并转化大肠杆菌,37℃下1mM IPTG诱导表达,得到了59kDa的特异性蛋白。超声破碎实验表明该重组蛋白主要以包涵体形式存在。重组蛋白经纯化浓缩后,注射家兔制得多克隆抗体。Western blot证明成功地制备了多克隆抗体,与表达的目的蛋白有特异性单一杂交带。优化pET-PdCYP51表达条件发现:在Rosetta（DE3）菌株中,30℃经1mM IPTG诱导获得了可溶性重组蛋白。根据柑桔绿霉菌CYP51跨膜结构的分析和三维空间结构的分析,重新构建了下列突变体,分别命名为pET-PdCYP51-18、pET-PdCYP51-66、pET-PdCYP51-244和p6P-PdCYP51-244,并在大肠杆菌中进行了表达。对pET-PdCYP51-18表达条件进行了优化,使其蛋白以可溶形式表达。同时构建了重组真核表达质粒pAUR-PdCYP51、p9K-PdCYP51和p9K-PdCYP51-244。3.根据CO差光谱法测定在Rosetta（DE3）中表达的PdCYP51重组蛋白活性。以柑桔绿霉菌微粒体和Rosetta（DE3）中表达的PdCYP51重组蛋白为材料,分析了商品化的烯唑醇、戊唑醇、三唑醇和三唑酮为代表的DMIs类杀真菌剂与CYP51的结合能力,分析了靶标酶活性、靶标酶纯度和靶标酶浓度对二者结合光谱的影响。结果表明,靶标酶活性和浓度是获得准确结合光谱的必要条件。根据米氏方程计算结合常数,计算得到烯唑醇、戊唑醇、三唑醇、三唑酮与靶标酶结合常数（Kd）,分别为0.12μM、0.32μM、0.64μM和0.99μM。该结果与其对柑桔绿霉菌生长抑制率EC₅₀显著相关,证明结合光谱与生物活性测定相结合的方法可作为一种简便可靠的DMIs类杀真菌剂筛选方法。4.利用建立的结合光谱法对合成的不同结构类型的化合物（XF系列、WJ系列和ZST系列）进行了结合光谱测定,结合生物测定的结果筛选出了一批结合能力强和杀菌效果好的新型抗真菌化合物。这些化合物均为含氮化合物,并且在直链的不同位置、咪唑环或三唑环上以卤素原子来取代。在XF系列化合物中,XF-22、XF50、XF-78、XF-100、XF-113、XF118和XF169与PdCYP51和PdCYP51-18的结合能力较强,同时生物活性测定效果较好。在WJ系列的化合物中,WJ7、WJ10、WJ11、WJ12、WJ15、WJ21、WJ22、WJ25和WJ26与PdCYP51的结合能力较强,结合常数均小于0.2μM;对柑桔绿霉菌的抑制率均为100%,有些化合物甚至在25mg/ml的浓度时其抑菌率仍在80%以上。ZST系列中的ZST1、ZST5和ZST9与PdCYP51的结合能力较强,抑制率与结合常数一致。5.利用计算机辅助设计手段（采用FLexX软件）将含氮的新型有机化合物与PdCYP51的活性中心卟啉基上的铁离子对接,通过模拟计算得知含氮化合物与PdCYP51活性中心最大的作用距离为3（?）,为设计高效、专一性强的新型抗真菌化合物提供了一种有效的手段。更多还原

【Abstract】 Green mold of citrus,caused by Penicillium digitatum Sacc,is the most serious postharvest disease of citrus.P.digitatum is the most harmful citrus fruit pathogen and infects the fruit during harvesting and processing.Up to date,14α-demethylation inhibitors（DMIs）are an important group of sterol biosynthesis inhibitors（SBIs）widely used in agriculture.Sterol 14α-demethylase（CYP51）is the only family of P450s,which is widely distributed in different biological kingdoms,found in animals,plants,fungi,yeast,lower eukaryotes and bacteria and considered to be the most ancient member of the superfamily.Cytochrome P450 14α-sterol demethylases are essential enzymes in sterol biosynthesis in eukaryotes.It removes the 14α-methyl group from sterol precursors such as lanosterol,obtusifoliol,dihydrolanosterol,and 24（28）-methylene-24,25-dihydrolanosterol.Inhibitors of CYP51 include triazole antifungal agents,such as fluconazole and itraconazole,drugs used in treatment of topical and systemic mycoses.Sterol biosynthesis is one of the general metabolic pathways present in the majority of eukaryotic cells and organisms.It leads to production of cholesterol in animals,ergosterol in fungi,and a variety of phytosterols in plants.The biosynthesis of sterol will lead to the damage or loss of structure and function of membrane via the inhibitation of 14α-demethylase,which will lead to the death of eukaryotes at last.In this study,sterol 14α-demethylase gene from P.digitatum was cloned and expressed,and the expressed product of the gene and the preliminary function were analyzed.Meanwhile,the 3D model was built and the fungicidal activities of some chemical entitles were screened according to the spectrophotometric method.The results are listed as follows:1.The P.digitatum CYP51 gene（PdCYP51）was cloned.Compared with the DNA sequence of P. digitatum strain PD5（accession no.AB030178）,there were four nucleotide changed（C1370→T, A1604→G,G1648→A and G1768→T）resulting in four different amino acids（P345L,E423G, G438R,V478L）.The cDNA sequence was registered in Genbank（accession no.DQ355161）. When compared to other amino acid sequences of CYP51 in Genbank,it indicated that PdCYP51 （ABC87815,Pd1）had 99%identities to that of P.digitatum strain PD5（BAB03658,Pd2）,86%to P.italicum（Q12664,Pi）,67%to Aspergillus fumigatus（AAF32372,Af）and Neosartorya fischeri （XP₀01267338,Nf）.All of the sequences contains SRS1（YxxF/LxxPxFGxxVxF/YD/a）,SRS4 （GQ/Hht/sS）and heine-binding protein sequence（FxxGxxxCxG）and four mutated amino acids all located outside the conserved regions.The four mutated amino acids have little relation with drug resistance according to the bioassay. 2.The three-dimensional（3D）model of PdCYP51 was built based on the structure template of le9x.pdb（Mycobacterium tuberculosis,MTCYP51）using FUGUE module of SYBYL7.0 program.The conserved amino acids of the activity cave were superimposed with the template. CYP51 is a kind of trans-membrane protein,displaying key roll in the biosynthesis of sterol. According to the analysis of hydrophobicity,signal peptide and transmembrane domain of PdCYP51,the N-terminus has 66 amino acids which stride across membrane twice,and the hydrophobic domain is mainly located in the transmembrane domain and the catalytic cave of the protein.The recombinant plasmid pET-PdCYP51 was constructed,and the 59kDa protein （PdCYP51）was obtained when induced by IPTG in E.coli BL21（DE3）strain at 37℃. Antibodies against purified PdCYP51 recombinant protein were raised in rabbit and western blotting was performed.It was indicated that the purified PdCYP51 had immunologic activity.3.The expressions of PdCYP51 were optimized under various conditions,and the soluble recombinant protein PdCYP51 was obtained in E.coli BL21（DE3）Rosetta at 25-30℃at last. According to the analysis of the transmemebrane structure and the homology modeling,various mutanted recombinant plasmids of pET-PdCYP51-18,pET-PdCYP51-66,pET-PdCYP51-244, p6P-PdCYP51-244 and p9K-PdCYP51-244.PdCYP51 were constructed and expressed in E.coli BL21（DE3）.The recombinant eukaryotic expression vector of pAUR-PdCYP51,p9K-PdCYP51 and p9K-PdCYP51-244 were also constructed.4.The activity of the soluble recombinant protein PdCYP51 was detected according to the CO difference spectra.The binding effect of the PdCYP51 with the representable antifungal agents, such as diniconazole,tebuconazole,triadimenol and triadimefon,were investigated through the binding spectra under various enzyme activity,enzyme purity and concentration.When compared with bioassay results,the binding spectra showed that the activity and proper concentration of the enzyme were necessary for obtaining accurate binding spectra.According to Michaelis-Menten equation,The Kd values of diniconazole,tebuconazole,triadimenol and triadimefon were 0.12μM、0.32μM、0.64μM and 0.99μM respectively,which significantly correlated to their EC₅₀ values on the growth of P.digitatum.These results indicated that the binding spectra of fungicide with sterol 14α-demethylase can serve as a reliable and quick method for the screening of novel fungicides.5.The activity of heterogenetic expressed PdCYP51 were investigated to build a binding-spectra method in screening the fungicidal activities of novel chemical entitles（serials XF、serials WJ and serials ZST）.According to the binding-spectra data and the bioassay results,some novel compounds with high activity were found,such as XF-22、XF-50、XF-78、XF-100、XF-113、 XF118 and XF169 of serials XF,and WJ7、WJ10、WJ11、WJ12、WJ15、WJ21、WJ22、WJ25 and WJ26 of serials WJ and ZST1、ZST5 and ZST9 of serials ZST.The Kd of WJ7、WJ10、WJ11、WJ12、WJ15、WJ21、WJ22、WJ25 and WJ26 were all less than 0.2μM,and the inhibition ratio all arrived at 100%on the growth of P.digitatum.Some chemical compounds even arrived at 80%at the concentration of 25mg/ml.6.The novel chemical entitles were docked to heme atom of the catalysis core of PdCYP51 by FLexX.In the binding model,the distance between N atom of chemical entitles and heme atom of the catalysis core of PdCYP51 was 3（?）obtained by computer aided design（CAD）.The typeⅡdifference spectral results also confirmed the reliability of the homology model,and showed that the selection criteria for the binding model of the compound were reliable,which provide an effective strategy to design novel compounds with high affectivity and specificity.更多还原