玉米纹枯病菌（Rhizoctonia solani AG-1-IA）β-1，4-内切纤维素酶的基因克隆表达及功能研究
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摘要
玉米纹枯病是世界上玉米产区广泛发生、危害严重的土传病害之一。在我国玉米纹枯病已成为玉米生产上的一种重要病害,且有逐年加重的趋势。其主要致病菌为立枯丝核菌(Rhizoctonia solani Kühn),属于丝核菌属,寄主范围十分广泛,能侵染水稻、玉米、大豆、大麦、小麦等40多个科200多种植物,引起纹枯、立枯等症状。玉米纹枯病菌可以产生多种细胞壁降解酶,如果胶酶、纤维素酶等,是病原物致病的主要因子。
本研究根据第45家族糖苷水解酶氨基酸保守区设计引物,从Rhizoctonia.solani AG-1-IA中克隆到两条45家族β-1,4-内切纤维素酶基因,分别命名为EG-1、EG-2。其中EG-1基因的DNA序列长1061bp,包含7段内含子区域,该基因cDNA和DNA在GenBank中的登录号分别为GU372731和GU372728;EG-2基因的DNA序列长1086bp,包含7段内含子区域,该基因cDNA和DNA在GenBank中的登录号分别为GU372730和GU372729。将EG-1、EG-2基因分别构建重组表达载体pPIC9K/EG-1和pPIC9K/EG-2,用限制性内切酶Sac I线性化后转化巴斯德毕赤酵母GS115。其中, pPIC9K/EG-1获得了酵母工程菌PEG-45,培养8d时表达量达到最高0.35mg/mL,SDS-PAGE确认纯化蛋白分子量约为28.05kDa,其最适反应pH为3.0,最适反应温度为45℃。pPIC9K/EG-2转化酵母,发酵后未检测到目的蛋白的表达。
将Rhizoctonia. solani菌丝块接种玉米叶片发现,菌丝接菌后侵染能力很强,24h后即表现明显病斑,RT-PCR证明菌丝在侵染过程中有β-1,4-内切纤维素酶基因EG-1、EG-2和EGIII的表达。通过改变Marcus诱导培养基的底物发现,β-1,4-内切纤维素酶的产生对底物存在依赖性,最适诱导底物为羧甲基纤维素钠CMC-Na。
将酵母工程菌株PEG-45表达的β-1,4-内切纤维素酶接种玉米叶片,发现在接种部位出现明显的病斑,且病斑沿叶脉扩展,以灭活酶液为对照,无病斑扩展。RT-PCR证明在发病过程中有防卫酶基因:病程相关蛋白(PR)、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)的表达。本实验从生理检测和基因表达两个方面对玉米纹枯病致病机制进行了初步研究,研究结果将为玉米纹枯病致病机制的深入研究、培育玉米纹枯病抗病新品种及研制防治玉米纹枯病的有效药剂奠定基础。
Corn sheath blight is a soil-borne disease in the Corn Belt around the world,which has become an important disease in corn production and increased gradually.The main pathogen of corn sheath blight is Rhizoctonia solani Kühn, which belonged to Rhizoctonia, having a widespread host range, including rice, corn, soybean, barley, wheat, and so on, causing sheath blight. Rhizoctonia.solani can produce many cell-wall degradation enzymes, such as Polygalacturonase (PG), cellulase(Cx), and become the main pathogenicity factor.
In this study, degenerate primers were designed on the conserved domain of other reported glycoside hydrolase family 45 protein. Through RT-PCR, 3’RACE PCR and 5’-TAIL PCR, twoβ-1,4-endo-celluase genes were cloned from Rhizoctonia solani AG-1-IA, named EG-1 and EG-2 respectively. The full-length of EG-1 DNA gene is 1061bp, containing seven introns, which accession number of cDNA and DNA in GenBank is GU372731 and GU372728 respectively; The full-length of EG-2 DNA gene is 1086bp, containing seven introns, which accession number of cDNA and DNA in GenBank is GU372730 and GU372729 respectively. The gene of EG -1 and EG-2 constructed recombination expression plasmid pPIC9K/EG-1 and pPIC9K/EG-2, linearized by restriction enzyme Sac I, then transformed to Pichia pastoris GS115. The recombinant P. pastoris PEG-45 was obtained from pPIC9K/EG-1, its expression level was up to 0.35mg/mL at the eight day. The molecular mass of a single band of the enzyme was estimated to be 28.05kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum reaction pH value of the expressed beta-1,4-endo-cellulase was 3.0, and the optimum reaction temperature was 45℃. The recombinant P. pastoris of pPIC9K/EG-2 was not detected target protein after fermentation.
we inoculated Rhizoctonia.solani to corn leaves, and found the hypha have a powerful infection ability. Through RT-PCR, we confirmed thatβ-1,4-endo-cellulase EG-1, EG-2 and EGIII can express in the process. By changing inductive substrates of Marcus medium, we found the optimum substrate forβ-1,4-endo-cellulase is CMC-Na.
we inoculated the corn leaves by purifiedβ-1,4-endo-cellulase from PEG-45, taken inactivated endocellulase as contrast, and found that there were obvious disease spots in vaccination sites and expanded along the veins while there were no expansion disease spot in the contrast. Through RT-PCR, we proved some defense genes expressed in the process, such as PR, POD, PAL. This study make a primary study from physiological detection and gene expression aspects for corn sheath blight, the research results will lay the foundation for further study the disease mechanism, foster new species and develop effective chemicals for sheath blight.
本研究根据第45家族糖苷水解酶氨基酸保守区设计引物,从Rhizoctonia.solani AG-1-IA中克隆到两条45家族β-1,4-内切纤维素酶基因,分别命名为EG-1、EG-2。其中EG-1基因的DNA序列长1061bp,包含7段内含子区域,该基因cDNA和DNA在GenBank中的登录号分别为GU372731和GU372728;EG-2基因的DNA序列长1086bp,包含7段内含子区域,该基因cDNA和DNA在GenBank中的登录号分别为GU372730和GU372729。将EG-1、EG-2基因分别构建重组表达载体pPIC9K/EG-1和pPIC9K/EG-2,用限制性内切酶Sac I线性化后转化巴斯德毕赤酵母GS115。其中, pPIC9K/EG-1获得了酵母工程菌PEG-45,培养8d时表达量达到最高0.35mg/mL,SDS-PAGE确认纯化蛋白分子量约为28.05kDa,其最适反应pH为3.0,最适反应温度为45℃。pPIC9K/EG-2转化酵母,发酵后未检测到目的蛋白的表达。
将Rhizoctonia. solani菌丝块接种玉米叶片发现,菌丝接菌后侵染能力很强,24h后即表现明显病斑,RT-PCR证明菌丝在侵染过程中有β-1,4-内切纤维素酶基因EG-1、EG-2和EGIII的表达。通过改变Marcus诱导培养基的底物发现,β-1,4-内切纤维素酶的产生对底物存在依赖性,最适诱导底物为羧甲基纤维素钠CMC-Na。
将酵母工程菌株PEG-45表达的β-1,4-内切纤维素酶接种玉米叶片,发现在接种部位出现明显的病斑,且病斑沿叶脉扩展,以灭活酶液为对照,无病斑扩展。RT-PCR证明在发病过程中有防卫酶基因:病程相关蛋白(PR)、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)的表达。本实验从生理检测和基因表达两个方面对玉米纹枯病致病机制进行了初步研究,研究结果将为玉米纹枯病致病机制的深入研究、培育玉米纹枯病抗病新品种及研制防治玉米纹枯病的有效药剂奠定基础。
Corn sheath blight is a soil-borne disease in the Corn Belt around the world,which has become an important disease in corn production and increased gradually.The main pathogen of corn sheath blight is Rhizoctonia solani Kühn, which belonged to Rhizoctonia, having a widespread host range, including rice, corn, soybean, barley, wheat, and so on, causing sheath blight. Rhizoctonia.solani can produce many cell-wall degradation enzymes, such as Polygalacturonase (PG), cellulase(Cx), and become the main pathogenicity factor.
In this study, degenerate primers were designed on the conserved domain of other reported glycoside hydrolase family 45 protein. Through RT-PCR, 3’RACE PCR and 5’-TAIL PCR, twoβ-1,4-endo-celluase genes were cloned from Rhizoctonia solani AG-1-IA, named EG-1 and EG-2 respectively. The full-length of EG-1 DNA gene is 1061bp, containing seven introns, which accession number of cDNA and DNA in GenBank is GU372731 and GU372728 respectively; The full-length of EG-2 DNA gene is 1086bp, containing seven introns, which accession number of cDNA and DNA in GenBank is GU372730 and GU372729 respectively. The gene of EG -1 and EG-2 constructed recombination expression plasmid pPIC9K/EG-1 and pPIC9K/EG-2, linearized by restriction enzyme Sac I, then transformed to Pichia pastoris GS115. The recombinant P. pastoris PEG-45 was obtained from pPIC9K/EG-1, its expression level was up to 0.35mg/mL at the eight day. The molecular mass of a single band of the enzyme was estimated to be 28.05kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum reaction pH value of the expressed beta-1,4-endo-cellulase was 3.0, and the optimum reaction temperature was 45℃. The recombinant P. pastoris of pPIC9K/EG-2 was not detected target protein after fermentation.
we inoculated Rhizoctonia.solani to corn leaves, and found the hypha have a powerful infection ability. Through RT-PCR, we confirmed thatβ-1,4-endo-cellulase EG-1, EG-2 and EGIII can express in the process. By changing inductive substrates of Marcus medium, we found the optimum substrate forβ-1,4-endo-cellulase is CMC-Na.
we inoculated the corn leaves by purifiedβ-1,4-endo-cellulase from PEG-45, taken inactivated endocellulase as contrast, and found that there were obvious disease spots in vaccination sites and expanded along the veins while there were no expansion disease spot in the contrast. Through RT-PCR, we proved some defense genes expressed in the process, such as PR, POD, PAL. This study make a primary study from physiological detection and gene expression aspects for corn sheath blight, the research results will lay the foundation for further study the disease mechanism, foster new species and develop effective chemicals for sheath blight.
引文
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