小麦叶片上潜伏期条锈菌的巢式PCR检测及小麦与条锈菌互作中相关激酶类基因的表达谱分析
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摘要
第一部分小麦叶片上潜伏期条锈菌的巢式PCR检测
由小麦条锈菌(Puccinia striiformis f. sp. Tritici)引起的小麦条锈病是世界范围内小麦生产上的一个重要病害,主要发生在气候凉爽潮湿的地区。在条锈菌夏孢子越冬时期,被病原菌侵染的小麦叶片在潜伏期不表现任何症状,一种快速可靠的检测方法有助于提早预测小麦条锈病的爆发,有效控制病害发生。
基于组织学,形态学等检测小麦条锈菌的传统方法费时耗力,还需要具备丰富的植物病理学知识。为加速并简化小麦条锈菌的检测方法,本研究建立了利用特异性高,灵敏度好的巢式PCR方法对小麦条锈菌进行检测。
1.引物特异性检测。根据小麦条锈菌的PSR序列设计特异引物Pst1-Pst2,利用小麦条锈菌7个不同的生理小种夏孢子及6种其他小麦病原菌DNA为模板检测其特异性。小麦条锈菌的7个生理小种都产生约470bp的目的条带,而其他小麦病原菌无产物。这说明,引物Pst1-Pst2具有小麦条锈菌特异性,而无小种特异性,适于小麦条锈菌的检测。
2.巢式PCR检测小麦条锈菌。第一轮PCR反应以依次稀释10倍的小麦条锈菌DNA为模板,利用引物Pst1-Pst2扩增,可检测到1pg小麦条锈菌。第二轮PCR反应以第一轮PCR产物为模板,利用内引物Nest1-Nest2扩增,检测灵敏度可达1fg。利用接种条锈菌的小麦叶片基因组DNA为模板,巢式PCR在接种24h后即可检测到病原菌的存在。巢式PCR特异性强,灵敏度高,在小麦条锈菌侵染早期即可检测到微量病原菌,为小麦条锈病的早期诊断提供了有利工具。
第二部分小麦与条锈菌互作中相关激酶类基因的表达谱分析
蛋白激酶是酶中的一个大家族,其可逆蛋白磷酸化作用在所有真核细胞基础功能调节中起重要作用,如细胞周期控制、能量代谢、细胞与细胞之间的通讯和介导与周围环境的复杂相互作用等。
为阐明小麦-小麦条锈菌互作过程中的一些分子事件及有关蛋白激酶的功能,本研究在本实验室已建立的小麦与小麦条锈菌互作非亲和/亲和组合cDNA文库及小麦与小麦条锈菌互作非亲和/亲和组合SSH文库中挑选蛋白激酶类基因,通过斑点杂交检测其在小麦与条锈菌互作中的表达趋势。选取表达水平有明显变化的蛋白激酶类基因,通过实时荧光定量PCR更精确的检测小麦条锈菌感染小麦过程中,小麦相关激酶类基因表达水平的变化。
1.斑点杂交检测小麦相关蛋白激酶类基因表达趋势。在实验室已建立的小麦与小麦条锈菌互作非亲和/亲和组合cDNA文库及小麦与小麦条锈菌互作非亲和/亲和组合SSH文库中挑选出94个蛋白激酶类基因的EST序列。对挑选出的93个EST质粒进行斑点杂交,检测在亲和及非亲和组合中,小麦在接种条锈菌0h,18h,24h,48h,72h后表达水平的变化。检测根据杂交结果,挑选出30个表达水平变化明显的激酶类基因。
2.相关激酶类基因的real-time PCR分析。以18SrRNA持家基因为内参,利用SYBR GreenⅠreal-time PCR方法分析了19个激酶类基因在亲和及非亲和组合中,小麦在接种条锈菌0h,12h,18h,24h,48h,72h,120h后表达水平的变化。在非亲和组合中,多数蛋白激酶类基因在条锈菌侵染前期表达量较高,尤其在接种后12h和24h,可能参与植物感受病原菌刺激的信号转导过程。另外一些表达量基本没有变化或者在亲和及非亲和组合中表达趋势基本一致,可能为组成型表达基因或与植物其他抗逆反应或生物学功能有关。
由于小麦基因组的复杂性,使小麦蛋白激酶的研究还属于零星的研究,远未能形成对蛋白激酶在各反应中的完整认识。小麦与条锈菌的互作是一个很复杂的过程,互作中小麦蛋白激酶的研究将为小麦的基础代谢、抗逆和抗病害的机理研究奠定基础。
PARTⅠDETECTION OF Puccinia striiformis IN LATEN INFECTED WHEAT LEAVES BY NESTED PCR
Stripe rust, caused by Puccinia striiformis f. sp. Tritici, is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. A rapid and reliable detection of the pathogen in latent infected wheat leaves during overwintering of the fungus in the dormant stage will contribute to determine the initial inoculum potential and thus to predict early outbreak and to improve effective management of the disease.
The identification of Puccinia striiformis based on morphology or physiological is rather labor-intensive and time-consuming. In order to accelerate and simplify the process of detection, a nested PCR assay was developed for specific and sensitive detection of P. striiformis in this research.
1. Detection of primers specificity.
Specific primers Pst1-Pst2 were designed according to a genome-specific sequence of P. striiformis. To evaluate the specificity of the primers, seven different isolates and races of P. striiformis as well as six other pathogens of wheat were tested. All isolates of P. striiformis yielded a distinct band of a fragment of 470bp, while using DNA of the other wheat pathogens as template no amplification product was detected. Primers internal to the species-specific outer primers Pst1-Pst2 were subsequently designed .
2. Detection of Puccinia striiformis in laten infected wheat leaves by nested PCR.
In nested PCR, with a 10-fold dilution series of template DNA, the limit amount of detection was 1pg DNA in the first PCR with the primers Pst1-Pst2. The second round PCR was then performed using amplified product from the first PCR as the template and Nest1-Nest2 as the primers. Amplification signal was detectable even when only 1fg of P. striiformis f. sp. Tritici DNA was used as the template for the first PCR. With nested PCR, the sensitivity of detection was enhanced by 100 folds. Using extracts from P. striiformis infected wheat leaves, the fungus could be determined in the leaves before symptom appeared. The assay provides a rapid and sensitive method for detection of P. striiformis in latent infected leaves of overwintering wheat plants.
PARTⅡEXPRESSION PROFILES ANALYSIS OF RELATED PROTEIN KINASE DURING THE INTERATION BETWEEN WHEAT AND STRIPE RUST
Protein kinase plays important roles in basic function regulation in all eukaryotic cells, such as regulation of cell cycles, energy metabolism, mediation of complex interactions between cells and surrounding environments and so on.
In order to demonstrate the molecular incidents and the functions of protein kinases during the comunication between wheat and stripe rust, protein kinase related genes were selected in the cDNA library and SSH library established during the interaction between wheat and stripe. The expression levels of all the selected genes were firstly detected by dot blot, then some of the genes interested in were analyzed by real-time PCR for more information about their expression.
1.Detection of kinase related genes expression by dot blot.
94 kinase related genes were selected and the expression levels of 0h, 18h, 24h, 48h and 72h after inocultion in compatible and incompatible groups were detected by dot blot . According to the dot blot results, 30 kinase related genes that shown obvious changes in expression levels were chosen.
2. Real-time PCR analysis of interesting kinase related genes.
In this research, SYBR GreenⅠreal-time PCR assay were established to detect the expression of kinase related genes during the process of stripe rust infecting wheat. 19 kinase related genes were analyzed by real-time PCR with the 18SrRNA as the control gene. For most kinase genes, the expression levels increased in the early stage after pathogen infection in incompatible reaction, which may be concerned with the signal transduction of pathogen infection. Some others kept the same expression levels during the infection process or shown the similar expression levels in both compatible and incompatible groups, which may be constitutely expressed engaged in other biological functions.
Nowdays, the wheat kinase was seldomly studied for the complicated genome of wheat, and the roles kinase played in basic function regulations haven’t been completely recognized. The interaction between wheat and stripe rust is an complicated process, and the study of wheat kinase would contribute to the research of wheat basic metabolism and the mechnism of disease resistance.
由小麦条锈菌(Puccinia striiformis f. sp. Tritici)引起的小麦条锈病是世界范围内小麦生产上的一个重要病害,主要发生在气候凉爽潮湿的地区。在条锈菌夏孢子越冬时期,被病原菌侵染的小麦叶片在潜伏期不表现任何症状,一种快速可靠的检测方法有助于提早预测小麦条锈病的爆发,有效控制病害发生。
基于组织学,形态学等检测小麦条锈菌的传统方法费时耗力,还需要具备丰富的植物病理学知识。为加速并简化小麦条锈菌的检测方法,本研究建立了利用特异性高,灵敏度好的巢式PCR方法对小麦条锈菌进行检测。
1.引物特异性检测。根据小麦条锈菌的PSR序列设计特异引物Pst1-Pst2,利用小麦条锈菌7个不同的生理小种夏孢子及6种其他小麦病原菌DNA为模板检测其特异性。小麦条锈菌的7个生理小种都产生约470bp的目的条带,而其他小麦病原菌无产物。这说明,引物Pst1-Pst2具有小麦条锈菌特异性,而无小种特异性,适于小麦条锈菌的检测。
2.巢式PCR检测小麦条锈菌。第一轮PCR反应以依次稀释10倍的小麦条锈菌DNA为模板,利用引物Pst1-Pst2扩增,可检测到1pg小麦条锈菌。第二轮PCR反应以第一轮PCR产物为模板,利用内引物Nest1-Nest2扩增,检测灵敏度可达1fg。利用接种条锈菌的小麦叶片基因组DNA为模板,巢式PCR在接种24h后即可检测到病原菌的存在。巢式PCR特异性强,灵敏度高,在小麦条锈菌侵染早期即可检测到微量病原菌,为小麦条锈病的早期诊断提供了有利工具。
第二部分小麦与条锈菌互作中相关激酶类基因的表达谱分析
蛋白激酶是酶中的一个大家族,其可逆蛋白磷酸化作用在所有真核细胞基础功能调节中起重要作用,如细胞周期控制、能量代谢、细胞与细胞之间的通讯和介导与周围环境的复杂相互作用等。
为阐明小麦-小麦条锈菌互作过程中的一些分子事件及有关蛋白激酶的功能,本研究在本实验室已建立的小麦与小麦条锈菌互作非亲和/亲和组合cDNA文库及小麦与小麦条锈菌互作非亲和/亲和组合SSH文库中挑选蛋白激酶类基因,通过斑点杂交检测其在小麦与条锈菌互作中的表达趋势。选取表达水平有明显变化的蛋白激酶类基因,通过实时荧光定量PCR更精确的检测小麦条锈菌感染小麦过程中,小麦相关激酶类基因表达水平的变化。
1.斑点杂交检测小麦相关蛋白激酶类基因表达趋势。在实验室已建立的小麦与小麦条锈菌互作非亲和/亲和组合cDNA文库及小麦与小麦条锈菌互作非亲和/亲和组合SSH文库中挑选出94个蛋白激酶类基因的EST序列。对挑选出的93个EST质粒进行斑点杂交,检测在亲和及非亲和组合中,小麦在接种条锈菌0h,18h,24h,48h,72h后表达水平的变化。检测根据杂交结果,挑选出30个表达水平变化明显的激酶类基因。
2.相关激酶类基因的real-time PCR分析。以18SrRNA持家基因为内参,利用SYBR GreenⅠreal-time PCR方法分析了19个激酶类基因在亲和及非亲和组合中,小麦在接种条锈菌0h,12h,18h,24h,48h,72h,120h后表达水平的变化。在非亲和组合中,多数蛋白激酶类基因在条锈菌侵染前期表达量较高,尤其在接种后12h和24h,可能参与植物感受病原菌刺激的信号转导过程。另外一些表达量基本没有变化或者在亲和及非亲和组合中表达趋势基本一致,可能为组成型表达基因或与植物其他抗逆反应或生物学功能有关。
由于小麦基因组的复杂性,使小麦蛋白激酶的研究还属于零星的研究,远未能形成对蛋白激酶在各反应中的完整认识。小麦与条锈菌的互作是一个很复杂的过程,互作中小麦蛋白激酶的研究将为小麦的基础代谢、抗逆和抗病害的机理研究奠定基础。
PARTⅠDETECTION OF Puccinia striiformis IN LATEN INFECTED WHEAT LEAVES BY NESTED PCR
Stripe rust, caused by Puccinia striiformis f. sp. Tritici, is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. A rapid and reliable detection of the pathogen in latent infected wheat leaves during overwintering of the fungus in the dormant stage will contribute to determine the initial inoculum potential and thus to predict early outbreak and to improve effective management of the disease.
The identification of Puccinia striiformis based on morphology or physiological is rather labor-intensive and time-consuming. In order to accelerate and simplify the process of detection, a nested PCR assay was developed for specific and sensitive detection of P. striiformis in this research.
1. Detection of primers specificity.
Specific primers Pst1-Pst2 were designed according to a genome-specific sequence of P. striiformis. To evaluate the specificity of the primers, seven different isolates and races of P. striiformis as well as six other pathogens of wheat were tested. All isolates of P. striiformis yielded a distinct band of a fragment of 470bp, while using DNA of the other wheat pathogens as template no amplification product was detected. Primers internal to the species-specific outer primers Pst1-Pst2 were subsequently designed .
2. Detection of Puccinia striiformis in laten infected wheat leaves by nested PCR.
In nested PCR, with a 10-fold dilution series of template DNA, the limit amount of detection was 1pg DNA in the first PCR with the primers Pst1-Pst2. The second round PCR was then performed using amplified product from the first PCR as the template and Nest1-Nest2 as the primers. Amplification signal was detectable even when only 1fg of P. striiformis f. sp. Tritici DNA was used as the template for the first PCR. With nested PCR, the sensitivity of detection was enhanced by 100 folds. Using extracts from P. striiformis infected wheat leaves, the fungus could be determined in the leaves before symptom appeared. The assay provides a rapid and sensitive method for detection of P. striiformis in latent infected leaves of overwintering wheat plants.
PARTⅡEXPRESSION PROFILES ANALYSIS OF RELATED PROTEIN KINASE DURING THE INTERATION BETWEEN WHEAT AND STRIPE RUST
Protein kinase plays important roles in basic function regulation in all eukaryotic cells, such as regulation of cell cycles, energy metabolism, mediation of complex interactions between cells and surrounding environments and so on.
In order to demonstrate the molecular incidents and the functions of protein kinases during the comunication between wheat and stripe rust, protein kinase related genes were selected in the cDNA library and SSH library established during the interaction between wheat and stripe. The expression levels of all the selected genes were firstly detected by dot blot, then some of the genes interested in were analyzed by real-time PCR for more information about their expression.
1.Detection of kinase related genes expression by dot blot.
94 kinase related genes were selected and the expression levels of 0h, 18h, 24h, 48h and 72h after inocultion in compatible and incompatible groups were detected by dot blot . According to the dot blot results, 30 kinase related genes that shown obvious changes in expression levels were chosen.
2. Real-time PCR analysis of interesting kinase related genes.
In this research, SYBR GreenⅠreal-time PCR assay were established to detect the expression of kinase related genes during the process of stripe rust infecting wheat. 19 kinase related genes were analyzed by real-time PCR with the 18SrRNA as the control gene. For most kinase genes, the expression levels increased in the early stage after pathogen infection in incompatible reaction, which may be concerned with the signal transduction of pathogen infection. Some others kept the same expression levels during the infection process or shown the similar expression levels in both compatible and incompatible groups, which may be constitutely expressed engaged in other biological functions.
Nowdays, the wheat kinase was seldomly studied for the complicated genome of wheat, and the roles kinase played in basic function regulations haven’t been completely recognized. The interaction between wheat and stripe rust is an complicated process, and the study of wheat kinase would contribute to the research of wheat basic metabolism and the mechnism of disease resistance.
引文
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