| Yellow rust,caused by Puccinia striiformis f.sp. tritici, is one of the most detrimental diseases of wheat, imposing great threat to wheat production in China. It has been proven that breeding and growing resistance cultivars is the most economic and environmental friendly way to control the disease. Therefore, cloning genes related to the interactions between wheat and stripe rust fungus, along with their expression pattern and functional analyses, could help to illuminate the mechanism of host-pathogen interactions and provide solid foundation for wheat resistance breeding and modification. In this study, 13 genes which might be related to the host-pathogen interactions in wheat cultivar Suwon 11 were cloned by combining homologous cloning, RACE, in-silicon cloning and database exploration. Some of them were studied by bioinformatics analysis, expression pattern and functional analysis. The main contents and conclusions are as follows:1. To study the roles of SNARE proteins in wheat-stripe rust interactions, several wehat SNARE genes were cloned and analysed through bioinformatics. Firstly, TaNpsn11 and TaNpsn13 were obtained by combining homologuous cloning and RACE. Meanwhile, TIGR wheat annotation database provided a source for SNARE gene cloning; after RT-PCR verification, 9 wheat SNARE genes were characterized, including three R-SNAREs: Tav-Snare13,TaVamp725 and TaSec22;two Qa-SNAREs:TaSyp132 and TaSyp22;two Qb-SNAREs:TaNpsn12 and TaBet1;one Qc-SNARE:TaSyp51 and one Qb+c-SNARE:TaSnap28.2. Expression pattern of plant specific SNARE family--NPSN family was studied by qRT-PCR. Differentitial expression of TaNpsn11~13 in wheat tissues revealed their diverse roles in differentiation and development of wheat organs. When challenged by stripe rust pathogen, TaNpsns were significantly up-regulated in incompatible interactions, indicating that they might play important roles in wheat's resistance to the pathogen. Among the four exogenous phytohormones, TaNpsn11 could be up-regulated by all phytohormones; TaNpsn12 was just up-regulated by salicylic acid (SA) and abscisic acid (ABA); TaNpsn13 was up-regulated by ABA and methy-jasmonate (MeJA) while down-regulated by ethylene (ETH). These results showed that TaNpsn11~13 might function in wheat resistance to stripe rust disease through different signaling pathways. TaNpsns could not be induced by wounding stress while significantly up-regualted by drought and low temperature. In response to high salinity,TaNpsn11 showed resistance while others showed sensitivity. At last, pRTL2-GFP-C1, a vector suitable for subcellular localisation of plant SNAREs, was successfully modified.3. A novel gene which might encode HIN1 in Suwon 11 was cloned by in-silico cloning. The gene, designated as TaHin1, contained a 642 bp open reading frame (ORF) in length, encoding 213 amino acid residues with a molecular weight 23.24 KDa and PI value 8.67. TaHIN1 might be a secreted protein, containing a signal peptide and a HIN1 domain. In the expression pattern analysis, TaHin1 expressed in wheat tissues without difference. Challenged by stripe rust fungus, TaHin1 was induced only in incompatible interaction, up-regualted by SA and MeJA while down-regulated by ABA. ETH could not induce the expression. TaHin1 was obviously up-regulated by various abiotic stresses. The results indicated that TaHin1 might facilitate wheat defense through SA and JA pathways, while ABA played a negative role. And it also played roles in wheat's response to abiotic stresses.3. A novel gene which might encode a wheat actin-depolymerizing factor (ADF)was obtained from TIGR wheat annotation database, designated as TaAdf. The full-length cDNA was 812 bp, contained a 642 bp ORF, encoding 138 amino acid residues with a molecular weight 16.10 KDa and PI value 5.65. Its genomic DNA contained three exons in which the first exon just contained the initiation codon ATG. It was localised on chromosome 6D by Chinese Spring Nullisomic-Tetrasomics. Subcellular localisation showed its distribution in the whole cell. A 16 KDa band was observed in protokaryotic expression, in accordance with predicated weight. In the analysis of expression patterns, TaAdf expressed differentially in wheat tissues, revealing its role in differentiation and development of wheat organs. Challenged by stripe rust pathogen, TaAdf expressed higher in compatible interaction than in incompatible one. Meanwhile it was mainly up-regualted by ABA, revealing that TaAdf might mediate the wheat susceptibility to stripe rust disease by controlling the stoma through ABA signaling pathway. Among abiotic stresses, drought and low temperature signaicantly up-regulated its expression, while high salinity and wounding had no effect. After the silience of TaAdf in wheat through VIGS, enhanced resistance to stripe rust fungus was observed. |
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