Exression Profile Of A Wheat β-1,3-Glucanase Gene Induced By Puccinia Striiformis F.Sp.Tritici And Primary Study Of Stripe Rust Gene Function Analysis System Development

Stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriks. (Pst), is a serious fungal disease of wheat worldwide. Epidemics of the disease cause huge yield losses and downgrading in grain quality. Great achievements have been made in epidemiology and disease control. Systemic research on the mechanism between wheat-Pst has been extensively carried out at the histology, cytology, and biochemical aspects. However, little was known about the molecular interaction between the host and microbe as well as the gene function of the fungi. In this present thesis, two major parts including three aspects related to Puccinia striiformis and the wheat-Pst interaction system were represented.1. Expression profile of a wheatβ-1,3-glucanase gene induced by PstTo investigate the role ofβ-1,3-glucanase (EC 3.2.1.39) in the resistance response of wheat (cv. Suwon 11) to stripe rust, a wheatβ-1,3-glucanase gene induced by Pst, designated as TaGlu, was cloned and characterized. TaGlu was predicted to encode a basic protein of 334 amino acids. Quantitative real-time PCR analysis revealed that the transcript of TaGlu was induced during both compatible and incompatible interactions with Pst, but the transcription level was much higher in the incompatible interaction than that in the compatible interaction. TaGlu also showed noticeable induction of gene expression in young green leaf tissues treated with salicylic acid, methyl jasmonate or ethylene. Immunogold labeling assay showed that the gold particles were localized mainly in the host cell wall and over the extrahaustorial matrix, and the labeling densities were found significantly higher in the incompatible interaction than those in the compatible interaction.2. Primary study of development of Pst avirulence gene screening systemA large quantity sequences of putative avirulence genes have been selected from different cDNA libraries of Pst based on their bioinformatics analysis. However, their functions were not identified due to the reason that gene function investigation system of Pst is unavailable. To overcome the limitations of existing methods to identify Pst avirulence genes, a strategy based on functional expression that exhibit HR-based resistance was repersented in this study. Four gram-negative bacteria endophytes (WS, WR, YR and PR) were isolated from four wheat adult plants. WS, which identified as Pseudomonas aeruginosa, was then used as a“microorganism-vector”to delivery and express Pst genes in plant leaves based on its growth characteristics and the the type III secretion system. A gateway expression vector containing Xanthomonas oryzae pv. oryzicola avirulence gene avrRxo1 was constructed and transformed into WS. After infiltration of the endophyte into the leaves of the cron cultivar which have related resistant gene Rxo1, an obvious HR was observed. The result indicated this system was able to delivery and express foreign gene in plant leaves. However, considering the problem of stability, the system still needs improvements before screening Pst avirulence genes.3. Primary study of Agrobacterium-mediated transformation of Puccinia striiformisThe biotrophic fungus, Pst, can not be cultured on the artificial medium and does not have known alternate host. It is difficult to use conventional genetic approaches to study the gene function. In order to facilitate the employment of molecular genetic techniques with Pst, the establishment of a transformation system for this fungus is required. Previous attempts of the Ps transformation were all based on particle bombardment.Here, we introduced DNA into urediospores by using the Agrobacterium-mediated transformation system. The vector p13Ef-HYG-RED was builded up by inserting stem rust Ef-1αpromoter controled hph and Red genes fragment into pCAMBIA1302. 4 co-cultivation methods were established and got certain collections of putative transformants. These results could be very important for stable transformation system development of rust fungus.