| Wheat powdery mildew disease,which is caused by Erysiphe graminis,is one of the most serious wheat diseases in China and many other countries of the world.Identifying more resistant genes to powdery mildew disease and studying the resistance mechanism become more and more important.Pm21 gene,which is located on the short arm of chromosome 6V of H.villosa,is a broad-spectrum and powerful powdery mildew resistance gene.Studying the transcription factors,signal transduction pathway and important defense genes involved in the resistance progress,on the one hand,will be valuable to investigate the mechanism of the broad-spectrum resistance;on the other hand, isolating the resistance gene will facilitate the improvement of the powdery mildew resistance level of wheat through genetic engineering.The inoculated and the uninoculated resistant H.villosa were used to study the resistance mechanism of Pm21 to the powdery mildew by microarray analysis using the Barleyl Genechip.Upregnlated expressed genes induced by Erysiphe graminis were identified by comparison of transcript profiling of inoculated sample with that of the uninoculated sample of resistant H.villosa,and these resistant related genes and other genes containing resistant domains were chosen for further analysis.In the present research,14 pairs of primers for RT-PCR were designed based on the sequences of upregulated probes which related to the resistance genes,and 7 pairs of them were found to be upregulated by inoculation with Erysiphe graminis in the resistant H.villosa,with different transcript profiling.Contig3875 and Contig16352 belong to the Myb transcription factors and zinc transporter protein(ZIP1),and they only expressed in 24h post-inoculation;While Contig5557,Contig13968 and Contig25420 are putative serine/threonine phosphatase, transmembrane protein and immediate-early fungal elicitor protein.The expression level of these 3 contigs in inoculated resistant H.villosa was higher than that in uninoculated resistant H.villosa,and then gradually decreased after inoculation.This was similar as Hv-S/TPK gene cloned by Cao(2005).The last two contigs:Contig8614 and Contig16386 are calmodulin-binding and disease resistance proteins with a wavelike expression level after inoculation.These result approved that the Barley1 genechip is reliable in screening the upregulated genes induced by Erysiphe graminis.According to the information of the upregulated genes,The Contig25420,which is a putative immediate-early fungal elicitor protein from Oryza sativa,was only upregulated in resistant H.villosa,and its expression level increased after inoculation.This suggests Contig25420 may relate to powdery mildew disease in H.villosa.In this report,a pair of primers(25420F/25420R) were designed based on the sequence of Contig25420,and used to screen the cDNA library from leaves of diploid H.villosa.A full-length cDNA clone was obtianed.Sequence analysis showed that this full-length clone contains 1692 nucleotides and its putative protein contains a conserved domain of modified RING finger domain,named U-Box,which has four strictly conserved amino acids:Cys,Met,Pro and Gly.This cDNA clone is highly homologous with CMPG genes in Oryza sativa,Arabidopsis thaliana,Nicotiana tabacum and Petroselinum crispum,so this clone was named as Hv-CMPG.Hv-CMPG has a new family ubiquitin-protein ligase(E3),U-Box,which determine the substrate specificity of Ubiquitination.For further research on this rapid elicited gene,cDNA of resistant H.villosa with non-inoculation,and 5min,15min,30min,60min,2hr,4hr,12h and 24h post-inoculation were used for semi-quantitative RT-PCR.The results showed that gene expression was reached its maximum levels at 30 min-60min after elicitor treatment.A transient expression system was used to investigate the function of Hv-CMPG gene in the resistant process of powdery mildew disease in H.villosa.Target gene was constructed into plant expression vector and transformed into leaf epidermal cell of a powdery mildew susceptible wheat variety by gene gun.GUS gene was co-transformed with the target gene to mark the transformed cells.After transformation,leaf surface was inoculated with powdery mildew conidiospores.Forty-eight hours after inoculation, penetrations of the fungi in transformed cells were observed to evaluate the effects of the Hv-CMPG gene on protection from the invasion of powdery mildew.The results implied that,Hv-CMPG gene,when transiently expressed in leaf epidermal cells of susceptible wheat variety,could partly inhibit the penetration of conidiospores,and to some extent increase the resistance of cells to powdery mildew.The fragments of Hv-CMPG have been constructed into BSMV-derived virus-induced gene silencing vectors,and would be used to further study whether this gene involve the Pm21-mediated resistance pathway in wheat.
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