Detection Of Puccinia Striiformis In Laten Infected Wheat Leaves By Nested PCR And Expression Profiles Analysis Of Protein Kinase During Interaction Between Wheat And Stripe Rust

PARTâ… DETECTION OF Puccinia striiformis IN LATEN INFECTED WHEAT LEAVES BY NESTED PCRStripe 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. PARTâ…¡EXPRESSION PROFILES ANALYSIS OF RELATED PROTEIN KINASE DURING THE INTERATION BETWEEN WHEAT AND STRIPE RUSTProtein 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 Greenâ… real-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.