结合光谱法在DMIs类杀真菌剂筛选中的应用

为建立一种快捷和准确的方法用于新型杀真菌剂的筛选,以外源表达的稻瘟菌羊毛甾醇14α-去甲基化酶为靶酶,以市售烯唑醇、戊唑醇、三唑醇、三唑酮为DMIs类杀真菌剂代表,分析了靶酶活性、靶酶纯度和靶酶浓度对二者结合光谱的影响,并与生物测试结果比较分析其可靠性。结果表明靶酶的高活性、无其他P450干扰和合适的靶酶浓度是获得准确结合光谱的必要条件。烯唑醇、戊唑醇、三唑醇、三唑酮与靶酶结合常数（K,/i>_d）分别为0.143μmol/L、0.24μmol/L、0.257μmol/L、0.307μmol/L,该结果与其对稻瘟菌生长抑制能力(120h-EC₅₀)显著相关,证明结合光谱法可作为一种简便可靠的DMIs类杀真菌剂筛选方法。     

稻瘟菌CYP51蛋白F螺旋区保守氨基酸残基与

【目的】研究稻瘟菌CYP51蛋白F螺旋区保守氨基酸残基与烯唑醇的相互作用机制,为稻瘟病菌新型高效特异杀菌剂的开发提供理论依据。【方法】设计稻瘟菌CYP51蛋白F螺旋区保守氨基酸残基突变体P222C、P222H、I223A、I223W、N224A、N224S,截去N端跨膜区36个氨基酸后,在大肠杆菌BL21(DE3) Rosetta菌株中过量表达,采用结合光谱法分析诱导蛋白对烯唑醇的结合能力。【结果】表达的目标蛋白均保持了对药物的结合能力,呈现出II型的结合光谱曲线。相对于野生型蛋白,突变体I223W和I223A对烯唑醇的结合常数Kd值基本不变,N224S、N224A、P222C的Kd值都略有增大,无显著性差异(P>0.05),但是,P222H的Kd值有了显著的增大(P<0.05),表明突变体P222H对烯唑醇的亲和能力显著降低。【结论】稻瘟菌CYP51 P222位点的疏水性与药物结合密切相关。     

黑杨水培中营养液抑菌处理研究

水培条件下进行快速繁殖是大量生产黑杨种苗的途径之一 ,营养液的抑菌处理是水培的一个重要方面。该研究进行了二硫氰甲烷、中生菌素、敌磺钠、链霉素、青霉素等五种杀细菌剂和多菌灵、福美双、三唑酮等三种杀真菌剂的抑菌试验 ,结果表明 ,杀细菌剂对微生物生长的抑制率要高于杀真菌剂 ,二硫氰甲烷和中生菌素能较好地抑制营养液中微生物的生长 ,浓度分别为 2 0mg/L和 1 5mg/L时 ,抑制率分别为 75 .60 %和78 94%。杀真菌剂和杀细菌剂复配对营养液中微生物生长的抑制表现出较好的加和作用 ,其中以中生菌素(1 5mg/L)与福美双 (5mg/L)复配的效果最好 ,抑制率为 87.48%。     

苹果套袋果实黑点病菌杀菌剂室内测定研究

为筛选有效抑制套袋苹果黑点病多种病原的杀菌剂,选择25%吡唑醚菌酯悬浮剂、250 g/L嘧菌酯悬浮剂、10%苯醚甲环唑水分散粒剂、430 g/L戊唑醇悬浮剂、30%氟菌唑可湿性粉剂和80%克菌丹水分散粒剂6种杀菌剂,采用菌丝生长速率法,对引起套袋苹果黑点病的6种主要病原菌进行室内抑菌活性测定,获得了对多种黑点病病原具有抑制活性的杀菌剂。嘧菌酯、吡唑醚菌酯、戊唑醇和苯醚甲环唑4种杀菌剂对6种病原具有较好的抑菌活性,EC₅₀均小于20μg/mL,具较好田间应用前景。结果说明甲氧基丙烯酸酯类杀菌剂嘧菌酯、吡唑醚菌酯和三唑类杀菌剂戊唑醇、苯醚甲环唑有望作为交替使用防治套袋苹果黑点病的候选农药。     

棉酚对海胆精子鞭毛轴丝及力蛋白(Dynein)ATP酶的抑制作用

本工作观测了不同浓度棉酚(10—80μmol/L)对海胆精子鞭毛轴丝及力蛋白ATP酶的影响,发现棉酚对之具有剂量依赖方式的抑制作用。 实验对比了几种ATP酶抑制剂对轴丝ATP酶的作用,结果表明它们之间差别很大。使酶活力下降一半所需的各种抑制剂浓度分别为:钒酸钠17μmol/L;棉酚36μmol/L;槲皮酮450μmol/L,DCCD[双环已基碳化二亚胺(Dicyclohexylcarbodiimide)]1,600μmol/L;乌本苷>2,000μmol/L。 本文对棉酚的杀精机理,抑制强度及其控制生育的意义进行了讨论。     

疏绵状嗜热丝孢菌原生质体的制备与再生

以疏绵状嗜热丝孢菌(Thermomyces lanuginosus)为供试菌株,研究了菌龄、酶的种类及浓度、酶解时间、酶解温度和稳渗剂对原生质体制备的影响及稳渗剂对原生质体再生的影响。结果表明,制备嗜热丝孢菌原生质体比较适宜的条件为:PDB液体培养基培养28 h,以0.7 mol/L NaCl为稳渗剂,0.15 mol/L的溶壁酶,30℃酶解4 h。原生质体再生以0.7 mol/L蔗糖作稳渗剂为最佳。     

Snai1 miRNA干扰质粒构建及胃癌细胞系稳定株筛选

目的：设计并构建针对Snai1的微小干扰核糖核酸（miRNA）,最终鉴定出有效干扰质粒并筛选稳定转染的胃癌细胞株SGC-7901。方法：设计并构建4对Snai1的pcDNATM6.2-GW/EmGFPmiR microRNA及1对无效对照microRNA干扰质粒。将干扰质粒用罗氏BD转染试剂转染胃癌细胞株SGC-7901,通过倒置荧光显微镜观察绿色荧光确定转染效率。分别用不同浓度l的杀稻瘟菌素作用于SGC-7901细胞,得到杀稻瘟菌素对SGC-7901细胞的筛选浓度。Westernblot检测4对干扰质粒、阴性对照质粒对snai1蛋白水平表达的影响。结果：测序表明,Snai1干扰序列及读码框完全正确,干扰质粒瞬时转染的SGC-7901细胞系在倒置荧光显微镜下观察绿色荧光达85%以上。杀稻瘟菌素对于SGC-7901细胞的筛选浓度为5μg/ml。Westernblot结果显示,干扰序列Mi-1对Snai1有较强的干扰效果。结论：成功构建了Snai1干扰真核表达载体,同时筛选出有效干扰质粒及稳定转染株,为进一步研究Snai1在胃癌中的作用奠定了基础。     

Snai1 miRNA干扰质粒构建及胃癌细胞系稳定株筛选

目的:设计并构建针对Snai1的微小干扰核糖核酸(miRNA),最终鉴定出有效干扰质粒并筛选稳定转染的胃癌细胞株SGC-7901。方法:设计并构建4对Snai1的pcDNATM6.2-GW/EmGFPmiR microRNA及1对无效对照microRNA干扰质粒。将干扰质粒用罗氏BD转染试剂转染胃癌细胞株SGC-7901,通过倒置荧光显微镜观察绿色荧光确定转染效率。分别用不同浓度l的杀稻瘟菌素作用于SGC-7901细胞,得到杀稻瘟菌素对SGC-7901细胞的筛选浓度。Westernblot检测4对干扰质粒、阴性对照质粒对snai1蛋白水平表达的影响。结果:测序表明,Snai1干扰序列及读码框完全正确,干扰质粒瞬时转染的SGC-7901细胞系在倒置荧光显微镜下观察绿色荧光达85%以上。杀稻瘟菌素对于SGC-7901细胞的筛选浓度为5μg/ml。Westernblot结果显示,干扰序列Mi-1对Snai1有较强的干扰效果。结论:成功构建了Snai1干扰真核表达载体,同时筛选出有效干扰质粒及稳定转染株,为进一步研究Snai1在胃癌中的作用奠定了基础。     

齿裂菌属和皮下盘菌属ISSR-PCR最佳反应条件的研究

在利用ISSR技术分析齿裂菌属和皮下盘菌属遗传多样性的研究中,为获得条带清晰、重复性好的ISSR扩增结果,对影响ISSR-PCR的条件进行了筛选,确定了此类菌物ISSR-PCR反应的最适宜条件:在15μLPCR反应体系中,10倍Taq酶缓冲液1.5μL,DNA模板8ng/μL,MgCl22.5mmol/L,dNTP0.15mmol/L,引物浓度0.4μmol/L,Taq酶1.00U,ddH2O9.0μL。最佳退火温度因不同的引物而定,最佳循环次数为35次。     

白腐真菌原生质体制备和再生条件的研究

对影响白腐真菌(5．776)原生质体制备和再生的条件：包括菌龄、水解酶液的种类及浓度、酶解温度、酶解时间、再生培养基的稳渗剂的选择进行了研究。通过单因素比较分析和正交实验得到最适合的白腐真菌原生质体制备和再生条件。结果表明：当菌龄为58h,采用1%纤维素酶和1%蜗牛酶(2：1)混合液,酶解温度30℃,酶解时间180min,用0．7mol/L氯化钠作渗透压稳压剂,白腐真菌(5．776)原生质体的形成数和再生率均比优化前大为提高,原生质体形成量为8．36×10~5个/mL,原生质体再生率为9．12%。     

Plant sterol 14 alpha-demethylase affinity for azole fungicides

Azole fungicides were thought to have much greater affinity for the fungal cytochrome P450 enzyme, sterol 14 alpha-demthylase (CYP51) than the plant orthologue. Using purified CYP51 from the plant Sorghum bicolor L Moenech, a direct comparison of the sensitivity to the fungicides triadimenol and tebuconazole has been carried out. S. bicolor CYP51 was purified to homogenity as determined by SDS--PAGE and specific heme content. Addition of the azole fungicides triadimenol and tebuconazole induced type II spectral changes, with saturation occurring at equimolar azole/P450 concentrations. Inhibition of reconstituted activities revealed only a threefold insensitivity of the plant CYP51 compared to a fungal CYP51, from the phytopathogen Ustilago maydis, as judged by IC(50) values. The implications for fungicide mode of action and application are discussed.     

Stereo-selective interaction of enantiomers of diniconazole, a fungicide, with purified P-450/14DM from yeast

R(-) isomer of diniconazole [S-3308L, (E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl)-1-+ ++penten-3-ol], a newly developed fungicide strongly inhibited lanosterol 14 alpha-demethylation catalyzed by a yeast cytochrome P-450 (P-450/14DM). On the other hand, S(+) isomer of diniconazole was a weaker inhibitor for P-450/14DM. The R(-) isomer combined with both ferric and ferrous P-450/14DM and interfered binding of CO to the cytochrome. The S(+) isomer also interacted with both forms of P-450/14DM but the absorption spectra of the S(+)-diniconazole complexes were different from those of the R(-)-diniconazole complexes. Furthermore, S(+) isomer did not significantly interfere the binding of CO to P-450/14DM. These observations suggest that P-450/14DM discriminates enantiomers of diniconazole and the R(-) isomer is more favorably fit for the active site of the cytochrome.     

Mechanism of binding of prothioconazole to Mycosphaerella graminicola CYP51 differs from that of other azole antifungals

Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml(-1) resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.     

Sensitivity of Magnaporthe grisea to the Sterol Demethylation Inhibitor Fungicide Propiconazole

Baseline sensitivity of Magnaporthe grisea to a sterol demethylation inhibitor (DMI) propiconazole was determined using 52 wild-type single-spore isolates. The 50% effective concentrations of these 52 isolates to propiconazole ranged from 0.145 to 1.446  μ g/ml. Among the 52 isolates, two (07–82 and 04–006) were hypersensitive to propiconazole. The propiconazole-hypersensitive (PHS) isolates were also hypersensitive to another DMI fungicide triadimefon, but not to a benzimidazole fungicide carbendazim. Compared with the propiconazole-sensitive (PS) isolates, the PHS isolates retained normal pathogenicity. Real-time PCR analysis showed that expression of cyp51 gene in the PHS isolates was not significantly different from that in the PS isolates. Analysis of DNA sequence of cyp51 gene showed that the PHS isolates 07–82 and 04–006 had an amino acid substitution at the codon position 234 and 450, respectively, where the amino acids were conserved in the CYP51 of other fungi, which indicated that the substitutions in CYP51 might be related to hypersensitivity of M. grisea to DMI fungicides.     

Non-sterol structural probes of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae

A number of non-sterol iron-liganding molecules were used to probe the active site of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae. Simple bi- and tricyclic aromatic amines were found to exhibit Type II binding spectra with the demethylase. Stereochemical and positional effects appear to play critical roles in the binding of these compounds to the demethylase. These compounds have been used to generate additional active-site structural information on this enzyme, currently a target for the development of new antifungal agents.     

Stereoselective interaction of an azole antifungal agent with its target, lanosterol 14 alpha-demethylase (cytochrome P-45014DM): a model study with stereoisomers of triadimenol and purified cytochrome P-45014DM from yeast

The effect of the four triadimenol stereoisomers on the purified yeast lanosterol 14 alpha-demethylase (cytochrome P-45014DM), the primary target of azole antifungal agents, was studied. (1S,2R)-Triadimenol was the most potent demethylase inhibitor and bound quantitatively to the enzyme below 0.05 microM. This isomer also interfered with the chemical reduction of cytochrome P-45014DM and the binding of CO to the cytochrome. The other isomers showed a lower inhibitory effect on the enzyme, and the order of activity was (1R,2R) greater than (1R,2S) greater than or equal to (1S,2S). Based on these findings and the reported preferred conformations for the triadimenol stereoisomers (Anderson, N.H. et al., Pestic. Sci. 15:310-316, 1984), it is predicted that orientation of the hydrophobic tert-butyl and p-chlorophenyl groups relative to the azole nitrogen is important to fit the antifungal agent in the active site of the demethylase.     

Integration of metabolomics and in vitro metabolism assays for investigating the stereoselective transformation of triadimefon in rainbow trout

Triadimefon is a systemic agricultural fungicide of the triazole class whose major metabolite, triadimenol, also a commercial fungicide, provides the majority of the actual fungicidal activity, i.e., inhibition of steroid demethylation. Both chemicals are chiral: triadimefon has one chiral center with two enantiomers while its enzymatic reduction to triadimenol produces a second chiral center and two diastereomers with two enantiomers each. All six stereoisomers of the two fungicides were separated from each other using a chiral BGB‐172 column on a GC‐MS system so as to follow stereospecificity in metabolism by rainbow trout hepatic microsomes. In these microsomes the S‐(+) enantiomer of triadimefon was transformed to triadimenol 27% faster than the R‐(?) enantiomer, forming the four triadimenol stereoisomers at rates different from each other. The most fungi‐toxic stereoisomer (1S,2R) was produced at the slowest rate; it was detectable after 8 h, but below the level of method quantitation. The triadimenol stereoisomer ratio pattern produced by the trout microsomes was very different from that of the commercial triadimenol standard, in which the most rat‐toxic pair of enantiomers (known as “Diastereomer A”) is about 85% of the total stereoisomer composition. The trout microsomes produced only about 4% of “Diastereomer A”. Complementary metabolomic studies with NMR showed that exposure of the separate triadimefon enantiomers and the racemate to rainbow trout for 48 h resulted in different metabolic profiles in the trout liver extracts, i.e., different endogenous metabolite patterns that indicated differences in effects of the two enantiomers. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Cloning of the CYP51 gene from the eyespot pathogen Tapesia yallundae indicates that resistance to the DMI fungicide prochloraz is not related to sequence changes in the gene encoding the target site enzyme

Resistance to sterol 14 alpha-demethylase inhibitor (DMI) fungicides has been correlated with mutations in the CYP51 gene encoding the target enzyme eburicol 14 alpha-demethylase. CYP51 was isolated from the eyespot pathogen Tapesia yallundae revealing a predicted 526-amino acid product exhibiting homology to other fungal CYP51s. CYP51 was sequenced from four field isolates sensitive or resistant to the DMI fungicide prochloraz and partially sequenced from two further isolates and eight progeny from a cross between prochloraz-sensitive and -resistant parents. Two alleles of the gene were detected termed CYP51-1 and CYP51-2. No correlation was found between sequence change and fungicide sensitivity. Therefore prochloraz resistance involved a mechanism other than mutation in the target site gene.     

Direct enantiomeric resolutions of chiral triazole pesticides by high-performance liquid chromatography

Cellulose-tris (3,5-dimethylphenylcarbamate; CDMPC) was synthesized and coated on aminopropylsilica to prepare chiral stationary phase (CSP). Normal-phase high-performance liquid chromatography (HPLC) methods for the resolutions of five chiral triazole pesticides, diniconazole, tebuconazole, hexaconazole, triadimefon and flutriafol, on the CSP were developed. Several operating parameters such as mobile phase composition, modifier and column temperature were studied for the optimization of the resolutions. Better separations were achieved using 2% iso-butanol for diniconazole, 2% ethanol for tebuconazole, 2% iso-propanol for hexaconazole, 1% n-butanol for triadimefon and 2% n-propanol for flutriafol as modifiers in n-hexane at 0 degrees C with the resolution factors (Rs) of 1.62, 1.66, 2.46, 1.68 and 1.98, respectively. Low temperature was better for the resolutions. Validation of the methods included linearity and precision.     

Sequence variation in the CYP51 gene of Blumeria graminis associated with resistance to sterol demethylase inhibiting fungicides

Resistance to sterol 14alpha-demethylase inhibiting fungicides (DMIs) has been correlated with mutations in the CYP51 gene, which encodes the target enzyme eburicol 14alpha-demethylase. To test the hypothesis that variation in the CYP51 gene explains variation for DMI sensitivity in barley and wheat powdery mildew species, this gene was sequenced from isolates of Blumeria graminis f.sp. hordei (Bgh) and f.sp. tritici (Bgt), respectively, which differed in their responses to DMIs in agricultural populations in the UK. Two single-nucleotide mutations in the CYP51 gene, which resulted in the amino acid substitutions Y136F and K147Q, were detected. K147Q is a novel mutation present only in Bgh isolates expressing very high levels of resistance. Sequence analysis of the CYP51 gene from the progeny of a cross between DMI-sensitive and resistant Bgh isolates showed that both mutations segregate with resistance, which is consistent with CYP51 controlling a major portion of DMI resistance. However, genetic analysis of resistance to the DMI triadimenol indicates that mutation of the CYP51 gene is not the only mechanism of resistance operating in B. graminis.