玉米大斑病菌2A型蛋白磷酸酶基因的克隆与功能分析
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摘要
由玉蜀黍大斑刚毛座腔菌(Setosphaeria turcica)引起的玉米大斑病是玉米生产上的重要病害之一,其发生和流行,不仅造成严重的经济损失,而且降低了玉米的品质。研究表明,许多植物病原真菌的生长、发育都受细胞跨膜信号转导途径的调控,如MAPK信号途径、cAMP信号途径及Ca2+信号途径等。研究信号转导途径对玉米大斑病菌生长和发育的调控作用,有利于深入了解病菌的发育和侵染寄主的分子机制,对研究病原真菌与寄主植物之间的互作以及植物病害防治也具有重要的理论意义。
PP2A是一种主要的丝/苏氨酸磷酸酶, PP2As已被证实通过调控某些基因的转录、RNA拼接及翻译过程,对不同生物的信号转导、新陈代谢以及细胞周期方面发挥重要的作用。
为了明确PP2A与玉米大斑病菌分生孢子萌发、附着胞形成及致病性之间的关系,实验利用PP2A特异性抑制剂—斑蝥素(Cantharidin)初步研究了玉米大斑病菌PP2A的功能。结果显示,Cantharidin对玉米大斑病菌菌丝生长及分生孢子的萌发均具有明显的抑制作用,且抑制率随抑制剂浓度升高而增强;并且可引起芽管的缩短和不规则膨大。此外,Cantharidin也可以导致病菌粗毒素的生物学活性明显降低。由此说明PP2A可以通过影响菌丝及芽管的极性生长,而调控玉米大斑病菌的菌丝及分生孢子发育;同时参与病菌的毒素合成。
根据已知植物病原真菌PP2A-C和PP2A-B基因的保守核苷酸序列设计简并性引物,以玉米大斑病菌基因组DNA为模板,扩增获得了PP2A-C和PP2A-B基因的同源片段。利用Genome Walking技术对所得的基因片段进行延伸,获得了1600 bp的全长PP2A-C基因和2425 bp PP2A-B基因片段。Southern杂交技术确定了PP2A-C基因在玉米大斑病菌基因组中以单拷贝形式存在。
为进一步研究基因功能,试验构建了PP2A-C基因的同源重组型基因敲除载体。对PP2A-C基因的基因敲除载体质粒利用PEG介导法转化玉米大斑病菌的原生质体,潮霉素筛选共获得了15个稳定的转化子。以潮霉素磷酸转移酶基因hph为探针,经Southern杂交验证,确定了转化子E为突变体。对E突变体进行表型分析,发现其生长速率和致病性明显降低。
以上结果表明玉米大斑病菌PP2A基因对病菌形态建成、分生孢子萌发、附着胞形成、致病性有调空作用。为进一步研究玉米大斑病菌的致病机理以及植物与病原物分子互作和开发新的植物病害防治方法奠定了基础。
Corn northern leaf blight, caused by Setosphaeria turcica, is an important disease in maize production and often causes serious economic losses. Studies have shown that growth and development of many plant pathogenic fungi is subject to cell transmembrane signal transduction, such as the MAPK signaling pathway, cAMP pathway and Ca2+ signaling pathways, etc. Research on the regulation mechanisms of signal transduction pathways involved in the growth and development of corn northern leaf pathogen, will help us understand the molecular mechanism of the development and infection of phytopathogens, and lay theoretical foundation for the study about the interaction mechanisms between host plants and pathogens and plant disease control.
Type 2A phosphoprotein phosphatase (PP2A) is a major Ser/Thr phosphatase. PP2As have been shown to play roles in the regulation of the cell cycle, signal transduction and metabolism by controlling gene transcription, RNA splicing, and translation in a variety of organisms.
In order to study the relationship between PP2A and conidia germination, appressorium formation and pathogenicity in S. turcica, cantharidin, the specific inhibitors of PP2A, was used to study the function of PP2A.
Cantharidin showed significant inhibition effects on hyphal growth and conidial germination. The higher was the inhibitor concentration, the lower was the germination ratio of spores. Cantharidin caused the germ tube shortening and cell swelling irregularly. In addition, cantharidin could also obviously decrease the biological activity of HT-toxin. It was indicated that PP2A might regulate hyphal and conidial germination by controlling the cell polarity, and also involved in the biosynthesis of HT-toxin in Setosphaeria turcica.
Degenerate oligonucleotide primers were designed based upon conserved nucleotide sequences of the known PP2A-C gene andPP2A-B gene in plant pathogenic fungi. We amplified their homologous fragments from S. turcica 01-23 genomic DNA. Then its flanking regions were obtained by Genome Walking. We got a PP2A-C full length DNA sequence of 1600 bp and a DNA homologous fragment of PP2A-B which is 2425 bp in length. The specific probe of PP2A-C was prepared to apply for Southern blotting. The results showed that PP2A-C gene gene only had single copy in genomic DNA of S. turcica.
To study the gene function deeply, the vector of homologous recombination of PP2A-C gene was constructed, and the protoplasts of S. turcica were transformed with it through PEG-mediated transformation system. 15 transformants were obtained after screening with hygromycin B. It was determined through Southern blotting that the transformant E was mutant. In contrast to the wild strain 01-23, the PP2A-C mutant showed different phenotypes at the same growth conditions. On PDA medium, filamentous growth of PP2A-C mutants was impaired. Pathogenicity assays showed that the pathogenicity of the mutants was decreased.
These results indicated that PP2A gene played an important role in the processes of conidium germination, appressorium formation and pathogenicity of S. turcica, which could lay a foundation for investigating pathogenic mechanism of S. turcica, molecular interaction between plant and pathogen, and new methods of disease prevention.
PP2A是一种主要的丝/苏氨酸磷酸酶, PP2As已被证实通过调控某些基因的转录、RNA拼接及翻译过程,对不同生物的信号转导、新陈代谢以及细胞周期方面发挥重要的作用。
为了明确PP2A与玉米大斑病菌分生孢子萌发、附着胞形成及致病性之间的关系,实验利用PP2A特异性抑制剂—斑蝥素(Cantharidin)初步研究了玉米大斑病菌PP2A的功能。结果显示,Cantharidin对玉米大斑病菌菌丝生长及分生孢子的萌发均具有明显的抑制作用,且抑制率随抑制剂浓度升高而增强;并且可引起芽管的缩短和不规则膨大。此外,Cantharidin也可以导致病菌粗毒素的生物学活性明显降低。由此说明PP2A可以通过影响菌丝及芽管的极性生长,而调控玉米大斑病菌的菌丝及分生孢子发育;同时参与病菌的毒素合成。
根据已知植物病原真菌PP2A-C和PP2A-B基因的保守核苷酸序列设计简并性引物,以玉米大斑病菌基因组DNA为模板,扩增获得了PP2A-C和PP2A-B基因的同源片段。利用Genome Walking技术对所得的基因片段进行延伸,获得了1600 bp的全长PP2A-C基因和2425 bp PP2A-B基因片段。Southern杂交技术确定了PP2A-C基因在玉米大斑病菌基因组中以单拷贝形式存在。
为进一步研究基因功能,试验构建了PP2A-C基因的同源重组型基因敲除载体。对PP2A-C基因的基因敲除载体质粒利用PEG介导法转化玉米大斑病菌的原生质体,潮霉素筛选共获得了15个稳定的转化子。以潮霉素磷酸转移酶基因hph为探针,经Southern杂交验证,确定了转化子E为突变体。对E突变体进行表型分析,发现其生长速率和致病性明显降低。
以上结果表明玉米大斑病菌PP2A基因对病菌形态建成、分生孢子萌发、附着胞形成、致病性有调空作用。为进一步研究玉米大斑病菌的致病机理以及植物与病原物分子互作和开发新的植物病害防治方法奠定了基础。
Corn northern leaf blight, caused by Setosphaeria turcica, is an important disease in maize production and often causes serious economic losses. Studies have shown that growth and development of many plant pathogenic fungi is subject to cell transmembrane signal transduction, such as the MAPK signaling pathway, cAMP pathway and Ca2+ signaling pathways, etc. Research on the regulation mechanisms of signal transduction pathways involved in the growth and development of corn northern leaf pathogen, will help us understand the molecular mechanism of the development and infection of phytopathogens, and lay theoretical foundation for the study about the interaction mechanisms between host plants and pathogens and plant disease control.
Type 2A phosphoprotein phosphatase (PP2A) is a major Ser/Thr phosphatase. PP2As have been shown to play roles in the regulation of the cell cycle, signal transduction and metabolism by controlling gene transcription, RNA splicing, and translation in a variety of organisms.
In order to study the relationship between PP2A and conidia germination, appressorium formation and pathogenicity in S. turcica, cantharidin, the specific inhibitors of PP2A, was used to study the function of PP2A.
Cantharidin showed significant inhibition effects on hyphal growth and conidial germination. The higher was the inhibitor concentration, the lower was the germination ratio of spores. Cantharidin caused the germ tube shortening and cell swelling irregularly. In addition, cantharidin could also obviously decrease the biological activity of HT-toxin. It was indicated that PP2A might regulate hyphal and conidial germination by controlling the cell polarity, and also involved in the biosynthesis of HT-toxin in Setosphaeria turcica.
Degenerate oligonucleotide primers were designed based upon conserved nucleotide sequences of the known PP2A-C gene andPP2A-B gene in plant pathogenic fungi. We amplified their homologous fragments from S. turcica 01-23 genomic DNA. Then its flanking regions were obtained by Genome Walking. We got a PP2A-C full length DNA sequence of 1600 bp and a DNA homologous fragment of PP2A-B which is 2425 bp in length. The specific probe of PP2A-C was prepared to apply for Southern blotting. The results showed that PP2A-C gene gene only had single copy in genomic DNA of S. turcica.
To study the gene function deeply, the vector of homologous recombination of PP2A-C gene was constructed, and the protoplasts of S. turcica were transformed with it through PEG-mediated transformation system. 15 transformants were obtained after screening with hygromycin B. It was determined through Southern blotting that the transformant E was mutant. In contrast to the wild strain 01-23, the PP2A-C mutant showed different phenotypes at the same growth conditions. On PDA medium, filamentous growth of PP2A-C mutants was impaired. Pathogenicity assays showed that the pathogenicity of the mutants was decreased.
These results indicated that PP2A gene played an important role in the processes of conidium germination, appressorium formation and pathogenicity of S. turcica, which could lay a foundation for investigating pathogenic mechanism of S. turcica, molecular interaction between plant and pathogen, and new methods of disease prevention.
引文
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[2]谷守琴.调控玉米大斑病菌生长发育和致病性的STK基因的克隆与功能分析[D].河北:河北农业大学博士学位论文,2007.
[3]范永山,曹志艳,谷守芹,等.不同诱导因素对玉米大斑病菌附着胞产生的影[J].中国农业科学,2004,37(5):769-772.
[4]孙大业,郭艳林.细胞信号系统[M].北京:科学出版社,1993.14.
[5] Lee Y H, Dean R A. cAMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea[J]. Plant Cell, 1993, 5 (6):693-700.
[6] Toda T, Cameron S, Sass P, et al. Cloning and characterization of BCY1,a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae[J]. Molecular Cell Biology, 1987, 7(4):1371-1377.
[7] Oliver B G, Panepinto J C, Fortwendel J R, et al. Cloning and expression of pkaC and pkaR,the genes encoding the cAMP-dependent protein kinase of Aspergillus fumigatus [J]. Mycopathologia, 2002, 154(2):85-91.
[8]刘卫军,沈瑛,丁健.蛋白磷酸酶2A的结构、功能和活性调节[J].生物化学与生物物理学报,2003,35(2):105-109.
[9] Mumby M, Walter G. Protein serine/threonine phosphatases:Structure, regulation, and functions in cell growth[J]. Physiol, 1993, 73:673-699.
[10] Holmes C, Boland M. Inhibitors of protein phosphatase1 and 2A: Two of the major serine/threonine protein phosphatases involved in cellular regulation[J]. Curr. Biol, 1993,3:934-943.
[11] Janssens V, Goris J. Protein phosphatase 2A: A highly regulated family of serine/ threonine phosphatases implicated in cell growth and signaling[J]. Biochem, 2001, 353:417-439.
[12] Schild A, Schmidt K, Lim Y A, et al. Altered levels of PP2A regulatory B/PR55 isoforms indicate role in neuronal differ-entiation[J]. Neuroscience, 2006, 24: 437-443.
[13] Adams D G, Coffee R L. Positive regulation ofRaf1-MEK1 /2-ERK1 /2 signaling by protein serine/threonine phosphatase 2A holoenzymes[J]. J BiolChem, 2005, 280: 42644-42654.
[14] Letourneux C, Rocher G, Porteu F. B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK[J]. EMBO Eur. Mol. Biol. Organ, 2006,25:727-738.
[15] Mumby M C, Walter G. Protein serine/threonine phosphatases: Structure, regulation,and functions in cell growth. Physiol[J]. 1993, 73:673-699.
[16] Dickman M, YardenO. Serine/threonine protein kinases and phosphatases in filamentous fungi[J]. Fungal Genet Biol, 1999, 26: 99-117.
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