Cloning And Functional Analysis Of Proline-Rich Protein Genes In Common Wheat

Fusarium Head Blight is a destructive fungal disease of small grain cereals and maize. In recent years, epidemic area of wheat scab has spreaded due to the fact of global warming as well as changes of farming systems. Since 1950s, intensive studies have been focused on pathogenicity and mechanisms of pathogenesis of Fusarium pathogen, screening of resistant resources, evaluation methods for host resistance, genetics analysis, QTL mapping and physiological and biochemistry mechanism of host resistance as well as improvement of disease resistance in wheat breeding. However, researches on gene cloning related to scab resistance and the molecular mechanism of FHB resistance are quite limited. The purpose of the present research is to screening, cloning and functional analysis of genes related to scab resistance using the FHB resistant vatiety wangshuibai and its fastnetron-induced susceptible mutants NAUH117 based on the microarray analysis.The main results obtained are as follows.1. The differentially expressed genes of the FHB fungal inoculated spikes between Wangshuibai and NAUH117 were screened using the Affymetrix Wheat Genome GeneChip. Among the differentially expressed genes, three proline-rich protein genes were down-regulated significantly in the mutant NAUH117 than in the Wangshuibai. It was proposed that the down-regulation of such kind of genes was necessary for the compatible interaction between the FHB pathogen and the host, and the stable expression of proline-rich protein genes may contribute to the resistance reaction of Wangshuibai to FHB pathogen.2. To obtain the putative FHB resistance related proline-rich protein genes in the Wanghuibai, primers were designed according to the sequence of the three down-regulated probes in the chip. By RT-PCR and RACE reactions, three PRP genes were cloned successfully, and were named as TaPRP1, TaPRP2 and TaPRP3. The cDNA sequence of the three genes were 945bp,932bp and 991bp in length, and the putative protein sequence were 171aa,171aa and 159aa in length respectively. TaPRP1, TaPRP2 and TaPRP3 were located in the chromosomes 3B,3A, and 3D by using the nulli-tetrasomic lines of Chinese Spring. Analysis of the amino acid sequence indicated that all the three gene contain N-terminal signal peptide, and they were supposed to belong to a class of secretion protein.3. Three recombinant vectors expressing the fused protein of PRP and GFP were constructed. Using the transient expression system, the subcellular localization of the expressed protein of the PRP genes in the onion epidermal cells were analyzed. The results showed that TaPRP1-GFP and TaPRP3-GFP fusion proteins were located in the nucleus, while TaPRP2-GFP fusion protein was located in the whole cell.4. To characterize the function of TaPRP in the FHB resistance pathway, the TaPRP3 gene was transformed into the callus of a moderate susceptible variety Yangmai 158 by gene bombardment. Totally,136 regenerated plants were obtained, in which 4 were identified as the positive transgenic plants. The preliminary evaluation result of the plants at To generation showed that the over expression of the TaPRP3 could increase the FHB resistance of Yangmai 158 to some extent.