CDNA-AFLP Analysis Of Differentially Expressed Genes In Adult-Plant Resistance To Stripe Rust With Wheat And Characterization Study Of Genes Related To The Interaction Between Wheat And Stripe Rust

Stripe rust, caused by Puccinia striiformis f. sp. tritici （Pst） is a the most damaging fungal disease of wheat （Triticum aestivum L.） in all over the world. It has been proved that breeding and growing resistant wheat cultivars is the most effective, economical and environmentally friendly way for controlling this disease. Therefore, molecular mechanism of synergism investigation of wheat plant and rust fungi is greatly helpful for revealing the pathogenic mechanism of the rust fungi and the resistant mechanism of the host and providing further information for selection and reasonable use of resistant wheat cultivars. In the present thesis, the main work related to Pst-wheat interaction system were represented.1. The cDNA-AFLP method is used to analyze the differentially expressed genes of adult plant resistant wheat cultivar’Xingzi9104’infected by PST pathotype CYR32.64 primer combinations had been used and the total number of about 34880 and 32320 differentially expressed transcript derived fragments （DE-TDFs） at seedling stage and adult-plant stage Xingzi9104 respectively were detected, of which 37 primer combinations were selected for producing reliable polymorphic bands. About 2529 DE-TDFs of seedling stage and 2201 DE-TDFs of adult stage were obtained, of which 304 and 330 were successfully purified. The total of 509 Unigenes were produced by cloning, sequencing and assembling analyses by Cap3 software, which consist of adult-plant DE-TDFs （aDE-TDFs） library of 259 unigenes and seedling DE-TDFs （sDE-TDFs） library of 250 unigenes. All the TDF were submitted to GenBank and got the accession Numbers.All the Unigens were compared to the NCBI non-redundant protein database using the BlastX program. Two libraries were manually classified as fourteen different categories separately based on functional annotations, besides the biggest parts of unclear classification and the unclassified （also as No hits） genes, the other functional genes including metabolism, energy, cell growth, transcription, protein synthesis, protein destination and storage, transporters, intracellular traffic, cell structure, cell signal transduction, disease/defence and transposon. Clarified the variations and proportion of differential genes expression during interaction of Pst-whaet （CYR32 and Xingzi9104）, and also laid a foundation for the new gene discovery.65 overlapped DE-TDFs between the two libraries with functions of energy, metabolism, disease/defence and the unclassified implied that these genes showed different expression patterns during interaction of Pst and wheat.2. Full-length cDNAs were obtained of five putative secreted protein genes from Pst haustoria using the PCR and 5’rapid amplification of cDNA ends （RACE）, and designated as PstSP2C7, PstSP11L10, PstSP11P10, PstSP6P1 and Pst15a23. Their transcripts were 1094 bp,837 bp,769 bp,1001 bp and 568 bp, respectively and encoding predicted proteins ranged from 89 to 203 amino acids of open reading frame （ORF） without significant similarities to any accessions in the GenBank protein database, but with some homologies to predicted proteins in P. graminis, the stem rust pathogen. Bioinformatics analysis indicated that all these five genes only had predicted secretary signal peptides in N-terminal and had no conserved functional motifs, trans-membrane helices and potential glycosyl-phosphatidylinositol （GPI） anchor sites. They were the protein which had signal peptide secreted way and located in outside of cells.3. qRT-PCR study showed that all these three genes of PstSP2C7, PstSP11L10and PstSP11P10 had the different expressed patterns in transcript levels in different developmental and infection stages of the pathogen. PstSP11L10 was expressed at the highest level in the infected leaves and much higher than in the urediniospores and germinated urediniospores, indicating that PstSP11L10 is presumably either in haustoria or the intercellular mycelium or both and more likely to act in the wheat-Pst interaction. PstSP2C7 had the lowest expression in the germinated urediniospores but had the highest expression level in urediniospores, Although not the highest, the PstSP2C7 expression in the infected leaves was not significantly lower than the level in urediniospores, which might be due to the fact that urediniospores were beginning to form in the infected leaves when leaf samples were taken to detect the gene transcripts. Though PstSP11P10 had the highest expression level in urediniospores, however, The PstSP11P10 expression in infected leaves was much lower than that in urediniospores, which indicated this gene’s expression maybe in suppression during the interaction. The different expression patterns of the three genes may indicate their different functions.4. A novel gene which might encode ADP/ATP carrier protein PstAAC9P1 from Pst haustoria was studied by 5’RACE with full length cDNA which contains open reading frame （ORF） of 134 amino acid residues with over 80% homology to many ADP/ATP carrier proteins form fungi like Aspergillus nidulans, Aspergillus fumigatus, Pyrenophora tritici-repentis,Verticillium albo-atrum, Gibberella zea, Botryotinia Fuckeliana and with 99% identity to P. graminis. gene PGTG₁4813.2. PstAAC9P1 had the highest transcription level in the infected leaves, indicated that this gene might play the activate role during the infection of Pst.