玉米黑粉菌CYP51的克隆表达及同杀菌剂分子结合光谱分析
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摘要
甾醇14α-去甲基化酶(也称P450 14-DM, CYP51)是唯一的广泛存在于细菌、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。它催化甾醇的14α-去甲基化反应。甾醇14α-去甲基化酶是真菌麦角甾醇合成过程的关键酶。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。麦角甾醇的缺失会损害细胞膜结构和功能，最终导致真菌死亡。目前农业上14α-去甲基化酶抑制剂(14α-demethylation inhibitors, 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。
本研究首先从玉米黑粉菌中克隆CYP51基因，并构建了重组表达质粒pET-YH、pGEXKG-YH，采用不同的IPTG诱导浓度，不同的诱导温度，不同的表达宿主菌表达，经SDS-PAGE分析玉米黑粉菌CYP51基因均没有得到有效的表达。通过对其氨基酸序列进行跨膜结构预测，结合SYBYL7.0同源模建分析结果，从而重新克隆了2个玉米黑粉菌CYP51基因，即N端截短了的突变体(分别截短20和35个氨基酸)，并构建了重组表达质粒(pETYH-20, pETYH-35, pGEXKGYH-20, pGEXKGYH-35)，最终pETYH-35重组表达质粒在E.coli BL21(DE3)中得到了大量表达，但是N端截短了20个氨基酸的突变体仍未能有效表达。
将得到的重组玉米黑粉菌CYP51蛋白初步纯化后测定蛋白质含量，并根据P450-CO差光谱的方法测定其活性及P450含量，得到其蛋白质含量2.92mg／mL，P450含量为3.763pmol／mg。重组玉米黑粉菌CYP51蛋白同四种标准杀菌剂和十种人工合成的三唑类XF系列新型小分子杀菌剂进行了结合光谱测定研究，根据重组CYP51和杀菌剂之间的结合光谱分型并采用米氏方程Hanes-Woolf作图法计算结合常数，得到初步结论：标准杀菌剂和XF系列杀菌剂相比，结合常数均较小，标准杀菌剂中三唑醇的结合常数最小，结合常数为0.057；XF系列杀菌剂中XF-113结合常数最小，结合常数为0.307。通过重组玉米黑粉菌CYP51蛋白和杀菌剂之间的结合常数，参考生物活性测定结果对杀菌剂杀菌效果进行初步的评估，并由此推断真菌CYP51同三唑类杀菌剂的分子构型之间的联系。通过生物信息学的手段对得到的结果进行统计分析以便为设计开发新型的具有更强结合作用和更高选择性DMIs类杀菌剂提供了理论依据和实验指导。本研究同时构建了重组真核酵母表达质粒pAUR123-YH，采用醋酸锂转化后筛选阳性转化子进行真核表达。植物病原真菌玉米黑粉菌CYP51的克隆和表达对研究CYP51结构和功能从而为研究植物病原真菌对现存DMIs类杀菌剂产生的抗性机制奠定了良好的基础，同时也为设计开发新型的杀菌剂提供了理论基础。
Sterol 14α-demethylase (other names: P450 14DM; CYP51) is the only member of the cytochrome P450 superfamily present in animals, plants, fungi ,bacteria and considered to be the most ancient member of the superfamily. In all cases it catalyzes a three-step reaction of sterol 14α-demethylation. In fungi, the complex sterol 14α-demethylation reaction presents one of the key steps in sterol biosynthesis, an essential metabolic pathway producing ergosterol. The action mode of 14α-demethylase inhibitors (DMIs) involves selective inhibition of the fungal sterol 14α-demethylase over the plant enzyme activity during treatment. Inhibition of sterol 14α-demethylation by DMI fungicides result in the depletion of ergosterol, accumulation of abnormal sterol intermediates. Consequently the growth of fungi was arrested.
This study reported that the Ustilago maydis CYP51 DNA were cloned and expressed in E. coli. The recombinant expression vectors (pETYH and pGEXKGYH) were constructed and the optimal expression conditions and different expression host which added the rare tRNAs were tried, but they could not express. Transmembrane structures of the Ustilago maydis CYP51 were analyzed by online software. Homology models of the CYP51 and the mutants were constructed to analyze the structures of among the N-terminal cleavage CYP51 protein by SYBYL7.0 software which was useful for further site-directed mutagenesis experiment. According to the analytics results, we constructed the recombinant expression vectors(pETYH-20、pGEXKGYH-20、pETYH-35、pGEXKGYH-35) and got the CYP51 mutants protein expressed in the pETYH-35, but other mutants could not express in E. coli.
U.maydis CYP51 protein was purified and determined its biological activity based on P450-CO spectrometry which used to determining the cytochrome P450 characteristic absorption peak. U.maydis CYP51 protein concentration was 2.92mg/mL, the P450 concentration was 3.763pmol/mg. Spectrometry experiments were taken out to compute the inhibition constants according to Michaelis-Menten equation Hanes-Woolf method. In the research, there were four standard antiseptics and ten artifical triazoles antiseptics named XF series. The results were that all the combine constants of standard antiseptics were smaller than the artifical antiseptics'. Triadimenol got the score 0.057 and it was the smallest in the standard antiseptics while XF-113 got the score 0.307 and it was the smallest in the artifical antiseptics. The combination of new synthetic small molecules fungicide bioassay results on microbicides for the initial assessment. Analying the cloning and expression of and U.maydis CYP51 with bioinformatics method will facilitate the further research on the structure and function of fungi CYP51, so as to design more specific DMI fungicides against fungi CYP51. Eukaryotic expression vector pAUR123-YH of Saccharomyces cerevisiae was constructed by the conventional lithium acetate method and colony PCR was used to identificating the positive clones. This study provides an important tool for detailed enzymological and mechanistic studies so as to develop stronger affinitive and more specific DMI fungicides and for studying the resistance mechanisms of phytopathogenic fungi to certain DMI fungicides.
本研究首先从玉米黑粉菌中克隆CYP51基因，并构建了重组表达质粒pET-YH、pGEXKG-YH，采用不同的IPTG诱导浓度，不同的诱导温度，不同的表达宿主菌表达，经SDS-PAGE分析玉米黑粉菌CYP51基因均没有得到有效的表达。通过对其氨基酸序列进行跨膜结构预测，结合SYBYL7.0同源模建分析结果，从而重新克隆了2个玉米黑粉菌CYP51基因，即N端截短了的突变体(分别截短20和35个氨基酸)，并构建了重组表达质粒(pETYH-20, pETYH-35, pGEXKGYH-20, pGEXKGYH-35)，最终pETYH-35重组表达质粒在E.coli BL21(DE3)中得到了大量表达，但是N端截短了20个氨基酸的突变体仍未能有效表达。
将得到的重组玉米黑粉菌CYP51蛋白初步纯化后测定蛋白质含量，并根据P450-CO差光谱的方法测定其活性及P450含量，得到其蛋白质含量2.92mg／mL，P450含量为3.763pmol／mg。重组玉米黑粉菌CYP51蛋白同四种标准杀菌剂和十种人工合成的三唑类XF系列新型小分子杀菌剂进行了结合光谱测定研究，根据重组CYP51和杀菌剂之间的结合光谱分型并采用米氏方程Hanes-Woolf作图法计算结合常数，得到初步结论：标准杀菌剂和XF系列杀菌剂相比，结合常数均较小，标准杀菌剂中三唑醇的结合常数最小，结合常数为0.057；XF系列杀菌剂中XF-113结合常数最小，结合常数为0.307。通过重组玉米黑粉菌CYP51蛋白和杀菌剂之间的结合常数，参考生物活性测定结果对杀菌剂杀菌效果进行初步的评估，并由此推断真菌CYP51同三唑类杀菌剂的分子构型之间的联系。通过生物信息学的手段对得到的结果进行统计分析以便为设计开发新型的具有更强结合作用和更高选择性DMIs类杀菌剂提供了理论依据和实验指导。本研究同时构建了重组真核酵母表达质粒pAUR123-YH，采用醋酸锂转化后筛选阳性转化子进行真核表达。植物病原真菌玉米黑粉菌CYP51的克隆和表达对研究CYP51结构和功能从而为研究植物病原真菌对现存DMIs类杀菌剂产生的抗性机制奠定了良好的基础，同时也为设计开发新型的杀菌剂提供了理论基础。
Sterol 14α-demethylase (other names: P450 14DM; CYP51) is the only member of the cytochrome P450 superfamily present in animals, plants, fungi ,bacteria and considered to be the most ancient member of the superfamily. In all cases it catalyzes a three-step reaction of sterol 14α-demethylation. In fungi, the complex sterol 14α-demethylation reaction presents one of the key steps in sterol biosynthesis, an essential metabolic pathway producing ergosterol. The action mode of 14α-demethylase inhibitors (DMIs) involves selective inhibition of the fungal sterol 14α-demethylase over the plant enzyme activity during treatment. Inhibition of sterol 14α-demethylation by DMI fungicides result in the depletion of ergosterol, accumulation of abnormal sterol intermediates. Consequently the growth of fungi was arrested.
This study reported that the Ustilago maydis CYP51 DNA were cloned and expressed in E. coli. The recombinant expression vectors (pETYH and pGEXKGYH) were constructed and the optimal expression conditions and different expression host which added the rare tRNAs were tried, but they could not express. Transmembrane structures of the Ustilago maydis CYP51 were analyzed by online software. Homology models of the CYP51 and the mutants were constructed to analyze the structures of among the N-terminal cleavage CYP51 protein by SYBYL7.0 software which was useful for further site-directed mutagenesis experiment. According to the analytics results, we constructed the recombinant expression vectors(pETYH-20、pGEXKGYH-20、pETYH-35、pGEXKGYH-35) and got the CYP51 mutants protein expressed in the pETYH-35, but other mutants could not express in E. coli.
U.maydis CYP51 protein was purified and determined its biological activity based on P450-CO spectrometry which used to determining the cytochrome P450 characteristic absorption peak. U.maydis CYP51 protein concentration was 2.92mg/mL, the P450 concentration was 3.763pmol/mg. Spectrometry experiments were taken out to compute the inhibition constants according to Michaelis-Menten equation Hanes-Woolf method. In the research, there were four standard antiseptics and ten artifical triazoles antiseptics named XF series. The results were that all the combine constants of standard antiseptics were smaller than the artifical antiseptics'. Triadimenol got the score 0.057 and it was the smallest in the standard antiseptics while XF-113 got the score 0.307 and it was the smallest in the artifical antiseptics. The combination of new synthetic small molecules fungicide bioassay results on microbicides for the initial assessment. Analying the cloning and expression of and U.maydis CYP51 with bioinformatics method will facilitate the further research on the structure and function of fungi CYP51, so as to design more specific DMI fungicides against fungi CYP51. Eukaryotic expression vector pAUR123-YH of Saccharomyces cerevisiae was constructed by the conventional lithium acetate method and colony PCR was used to identificating the positive clones. This study provides an important tool for detailed enzymological and mechanistic studies so as to develop stronger affinitive and more specific DMI fungicides and for studying the resistance mechanisms of phytopathogenic fungi to certain DMI fungicides.
引文
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