PKA催化亚基调控玉米大斑病菌致病性的机制研究
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摘要
玉米大斑病菌(Setosphaeria turcica)引起的玉米大斑病(Northern Corn Leaf Blight)是威胁玉米生产的一种重要叶部真菌病害,常给玉米生产造成重大经济损失。研究表明,cAMP信号转导途径能够调控病原真菌的生长和发育,并与致病性有关。cAMP途径主要组分包括蛋白激酶A(Protein kinase A,PKA)和腺苷酸环化酶(Adenylyl cyclase,AC)。蛋白激酶A是该途径下游的主要效应分子,与一些病原真菌的致病性有直接关系。
本研究利用RACE技术对前期所得的玉米大斑病菌的cAMP依赖性蛋白激酶A催化亚基基因(PKA-c)片段进行延伸,获得了PKA-c全长基因。生物信息学分析表明,PKA-c基因与小麦黄斑叶枯病菌(Pyrenophora tritici-repentis)和烟草赤星病菌(Alternaria alternate)的相似性高达97%和92%;比对cDNA和DNA序列后发现,该基因有两个内含子,开放阅读框大小为1524 bp,编码507个氨基酸。本试验还在mRNA水平上研究了PKA-c的表达规律,发现PKA-c参与碳源物质的利用,且受山梨醇高渗胁迫的影响,此外,PKA-c还参与高渗环境下病菌的钠盐渗透胁迫反应,研究结果为进一步明确cAMP信号转导途径在玉米大斑病菌致病过程中的作用提供了理论依据。
为进一步明确PKA-c基因与玉米大斑病菌致病性的关系,本试验对玉米大斑病菌的PKA-c基因进行了RNA干扰研究。将PKA-c基因的RNAi载体质粒利用PEG介导法转化玉米大斑病菌的原生质体,潮霉素和PCR筛选共获得了5个转化子。通过半定量RT-PCR以及Northern blotting方法,验证转化子M3、M5、M9为突变体。对基因沉默突变体进行生物学性状分析,发现其菌丝生长速度高于野生型,但分生孢子产生能力下降,萌发所需时间变长;转化子的致病性较野生型明显降低,但两者产生HT-粗毒素的能力没有明显差别。试验还发现,在PKA-c基因表达量下降的同时,伴随着黑色素转录因子、木聚糖酶基因和微管蛋白基因表达量的下降。经对菌株产生的木聚糖酶酶活进行了测定,发现突变体的酶活比野生型低。由此得出结论,PKA-c基因参与调控玉米大斑病菌分生孢子的产生及萌发、参与黑色素的合成及木聚糖酶的合成,但与毒素的产生无关。
Setosphaeria turcica, an important fungal pathogen, is the threat of corn production. Lots of researches found that cAMP signal transduction pathway could regulate the growth, development and pathogenesis of pathogenic fungi. The components of cAMP pathway include protein kinase A and adenylyl cyclase, and protein kinase A is the main effector of cAMP signal transduction pathway, which is directly related to the pathogenecity of pathogenic fungi.
RACE technology was used to obtain the full length sequence of cAMP dependence protein kinases A catalytic subunit gene (PKA-c). The bioinformatic analysis revealed that the deduced amino acid sequence of the PKA-c owned 97% identity with Pyrenophora tritici-repentis (XP_001939935.1) and 92% identity with Alternaria alternate (ABY83137.1).
The cDNA and DNA sequences of PKA-c were compared by DNAMAN, the ORF (open reading frame), which was interrupted by two introns, was 1524 bp long, and encoded a protein with 507 amino acid residues. The expression of this gene was analyzed on mRNA level; we found PKA-c involving in the utilization for carbon source, influenced by the osmotic stress from sorbitol, and participate in stress reaction of plant pathogenic fungi under hypertonic environment by sodium salt. This research laid a foundation for the functional analysis about cAMP signal transduction pathway in phytopathogenic fungi.
RNAi (RNA interference) technology has been widely applied in the study of gene function due to its efficiency and economy. To further define the relationship between PKA-c gene and pathogenesis of S. turcica, we used RNAi (knockdown) as a tool for targeting endogenous gene PKA-c in S.turcica. PEG mediated transformation of PKA-c RNAi plasmid into protoplasts of S.turcica. The transformants were screened by hygromycin B and PCR (polymerase chain reaction) with specific primers corresponding to hygromycin. Three mutants M3, M5, M9 were obtained by semi-quantitative RT-PCR and Nouthern bloting analysis performed. Phenotypic analysis of these mutants showed that the extend of mutant hyphae grew faster than the wild isolate, conidia production was reduced, germination required longer periods of time and their pathogenicity significantly reduced, but crude HT-toxin activity between mutant and wild type were not strikingly difference. The decreased expression of PKA-c gene accompanied with melanin transcription factor, xylanase gene and tubulin gene expression decrease, while the xylanase of wild strain and mutant strain were extracted, enzyme activity was measured and the result showed that mutant had low activity. Therefore, It was concluded that PKA-c gene was involved in regulating conidia production and germination of S. turcica, and related to synthesis of melanin and activity of xylanase not directly relate to the activity of HT-toxin.
本研究利用RACE技术对前期所得的玉米大斑病菌的cAMP依赖性蛋白激酶A催化亚基基因(PKA-c)片段进行延伸,获得了PKA-c全长基因。生物信息学分析表明,PKA-c基因与小麦黄斑叶枯病菌(Pyrenophora tritici-repentis)和烟草赤星病菌(Alternaria alternate)的相似性高达97%和92%;比对cDNA和DNA序列后发现,该基因有两个内含子,开放阅读框大小为1524 bp,编码507个氨基酸。本试验还在mRNA水平上研究了PKA-c的表达规律,发现PKA-c参与碳源物质的利用,且受山梨醇高渗胁迫的影响,此外,PKA-c还参与高渗环境下病菌的钠盐渗透胁迫反应,研究结果为进一步明确cAMP信号转导途径在玉米大斑病菌致病过程中的作用提供了理论依据。
为进一步明确PKA-c基因与玉米大斑病菌致病性的关系,本试验对玉米大斑病菌的PKA-c基因进行了RNA干扰研究。将PKA-c基因的RNAi载体质粒利用PEG介导法转化玉米大斑病菌的原生质体,潮霉素和PCR筛选共获得了5个转化子。通过半定量RT-PCR以及Northern blotting方法,验证转化子M3、M5、M9为突变体。对基因沉默突变体进行生物学性状分析,发现其菌丝生长速度高于野生型,但分生孢子产生能力下降,萌发所需时间变长;转化子的致病性较野生型明显降低,但两者产生HT-粗毒素的能力没有明显差别。试验还发现,在PKA-c基因表达量下降的同时,伴随着黑色素转录因子、木聚糖酶基因和微管蛋白基因表达量的下降。经对菌株产生的木聚糖酶酶活进行了测定,发现突变体的酶活比野生型低。由此得出结论,PKA-c基因参与调控玉米大斑病菌分生孢子的产生及萌发、参与黑色素的合成及木聚糖酶的合成,但与毒素的产生无关。
Setosphaeria turcica, an important fungal pathogen, is the threat of corn production. Lots of researches found that cAMP signal transduction pathway could regulate the growth, development and pathogenesis of pathogenic fungi. The components of cAMP pathway include protein kinase A and adenylyl cyclase, and protein kinase A is the main effector of cAMP signal transduction pathway, which is directly related to the pathogenecity of pathogenic fungi.
RACE technology was used to obtain the full length sequence of cAMP dependence protein kinases A catalytic subunit gene (PKA-c). The bioinformatic analysis revealed that the deduced amino acid sequence of the PKA-c owned 97% identity with Pyrenophora tritici-repentis (XP_001939935.1) and 92% identity with Alternaria alternate (ABY83137.1).
The cDNA and DNA sequences of PKA-c were compared by DNAMAN, the ORF (open reading frame), which was interrupted by two introns, was 1524 bp long, and encoded a protein with 507 amino acid residues. The expression of this gene was analyzed on mRNA level; we found PKA-c involving in the utilization for carbon source, influenced by the osmotic stress from sorbitol, and participate in stress reaction of plant pathogenic fungi under hypertonic environment by sodium salt. This research laid a foundation for the functional analysis about cAMP signal transduction pathway in phytopathogenic fungi.
RNAi (RNA interference) technology has been widely applied in the study of gene function due to its efficiency and economy. To further define the relationship between PKA-c gene and pathogenesis of S. turcica, we used RNAi (knockdown) as a tool for targeting endogenous gene PKA-c in S.turcica. PEG mediated transformation of PKA-c RNAi plasmid into protoplasts of S.turcica. The transformants were screened by hygromycin B and PCR (polymerase chain reaction) with specific primers corresponding to hygromycin. Three mutants M3, M5, M9 were obtained by semi-quantitative RT-PCR and Nouthern bloting analysis performed. Phenotypic analysis of these mutants showed that the extend of mutant hyphae grew faster than the wild isolate, conidia production was reduced, germination required longer periods of time and their pathogenicity significantly reduced, but crude HT-toxin activity between mutant and wild type were not strikingly difference. The decreased expression of PKA-c gene accompanied with melanin transcription factor, xylanase gene and tubulin gene expression decrease, while the xylanase of wild strain and mutant strain were extracted, enzyme activity was measured and the result showed that mutant had low activity. Therefore, It was concluded that PKA-c gene was involved in regulating conidia production and germination of S. turcica, and related to synthesis of melanin and activity of xylanase not directly relate to the activity of HT-toxin.
引文
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[3]Dong J G, Fan Y S, Gui X M, et al. Geographic distribution and genetic analysis of physiological races of Setosphaeria turcica in Northern China[J]. American Journal of Agricultural and Biological Sciences, 2008, 3(1): 389-398.
[4]董金皋.农业植物病理学(北方本)[M].北京:中国农业出版社, 2001.
[5]侯晓强,范永山,董金皋,等.玉米大斑病菌有性杂交F1代菌株的生理小种鉴定和AFLP分析[J].植物保护学报, 2006, 33(3): 257-262.
[6]Dean R A. Signal pathways and appressorium morphogenesis[J]. Annual Review of Phytopathology, 1997, 35: 211-234.
[7]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响[J].中国农业科学, 2004, 37 (5): 769-772.
[8]Neves S R, Ram P T, Iyengar R. G protein pathways[J]. Science, 2002, 296: 1636-1639.
[9]姜俏,林琳,汪天虹.丝状真菌细胞凋亡研究进展[J].微生物学报, 2008, 48(4): 551-555.
[10]Choi W B, Kang S H, Lee Y W, et al. cAMP restores appressorium formation inhibited by polyamines in Magnaporthe grisea[J]. Phytopathology 1998, 88(1): 58-62.
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