| Powdery mildew,caused by Blumeria graminis f.sp.tritic(Bgt),is a destructive foliar disease of common wheat(Triticum aestivum),causes substantial yield losses in China and other parts of the world.Microarray is an effective method for analyzing differentially expressed genes.It can acquired the gene expression information at the transcriptional level quickly,effectively and in high throughput,so the Microarray technology is widely used in the study of the gene expression of wheat.Using GeneChip analysis method to research the metabolic pathway,analyze the signal transduction mechanisms of wheat under the stress of powdery mildew in whole genome level and then study its resistance mechanism,screen resistance related genes to powdery mildew.This is of great significance to understand the genetic basis of wheat resistance to powdery mildew and cultivate lasting disease-resistant varieties.Germplasm Edinburgh-b(introduced from Edinburgh in United Kingdom)displays immune to powdery mildew,it has a high use value in breeding,so it’s necessary to study the resistance mechanism.In the prent thesis,genechip analyses were employed for expression profiling in response to Bgt infection in the resistance germplasm Edinburgh-b.The main results obtained from this study are as follow:1.Screening the differential expressed genes of Edinburgh-b and Aikang58Leaf samples of Edinburgh-b and Aikang58 infected by Bgt were collected.The total RNA isolated from uninoculated leaves as the control,and total RNAs isolated from five samples after inoculated was pooled as the test.Data obtained from hybridization of the samples cRNAs crossed with wheat genechip were normalized.Probe sets were filtered for the signal value≥7.0 eithor in the test or in the control,and a fold-change threshold>2.0 or<0.5 as differentially expression.Comprison signal value of inoculated sample with that of the uninoculated sample of Edinburgh-b,about 5,835 probes were differentially expressed,among them,2,801 were up regulated,3,034 were down regulated.While in Aikang 58,about 4,447 probes were differentially expressed,among them,2,140 were up regulated,2,307 were down regulated.2.Verification of gene expression by qRT-PCRTo validate further the microarray data,we performed quantitative RT-PCR(qRT-PCR)on 17 differentially expressed genes before and after the infection in Edinburgh-b.Among them,15 genes were up regulated and the other 2 were down regulated.Reverse transcribed total RNA isolated from the uninoculated Edinburgh-b leaves and the mixture total RNAs isolated from inoculated Edinburgh-b five samples into cDNA as templates,using SYBR Green I fluorescent dye detection and 2-△△Ct to study the relative quantification,the results showed that the expression patterns of the selected genes were in good agreement with those of the microarray analysis.Then reverse transcribed total RNA isolated from the Edinburgh-b leaves at 0,6,12,24,48,72 hour post inoculation into cDNA as templates,analyze the gene expression patterns at 6 time points,the resultsshowed that some genes expression are differential at different time point,but the gene expression patterns are in good agreement with those of mixed samples,so use mixed samples can improve the screening efficiency.Moreover,24 hour post inoculation maybe a critical time point for defence response to powdery mildew in Edinburgh-b.3.Analysis the gene expression profiling of Edinburgh-b infected by BgtComprison of transcript profiling of inoculated sample with that of the uninoculated sample of Edinburgh-b,the differential expressed genes are mainly including enzymes related to metabolism,transcription factors,pathogen related proteins,defense genes,signal transduction components and so on.Pathway analysis showed that differentially expressed genes involved in the phenypropanoid biosynthesis,jasmonic acid(JA)biosynthesis,active oxygen metabolism and other metabolic processes.GO classification obtained some resistance-related biological processes,response to stimulus accounted for 11.10%,the immune system process and deaths accounted for 2.29%and 1.28%,respectively.Comprehensive analysis the expression profiling of Edinburgh-b and Aikang58 infected by Bgt,the results showed that salicytic acid(SA)and JA signal pathway involved in the resistance defence pathway in Edinburgh-b,JA signal pathway involved in the resistance defence pathway in Aikang58.JA signal pathway involved in both Edinburgh-b and Aikang58,only salicylic acid signaling pathway is difference,So SA signal signal pathway maybe the most effective in the resistance defence pathway of Edinburgh-b,while JA signal pathway plays a assist role. |
