| Wheat powdery mildew disease, which is caused by Blumeria graminis f. sp. tritici. 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.Hv-S/TPK has been cloned as a candidate gene of Pm21 through microarray analysis using the Barleyl Genechip. which is upregulated by Bgt. It can improve the disease resistance after transformed into the susceptible wheat Yangmail58. Although 6AS.6BS and 6DS of common wheat all contain the orthologous genes of Hv-S/TPK. the common wheat is still susceptible to powdery mildew. To study the resistant mechanism of Hv-S/TPK, two orthologous genes of Hv-S/TPK from 6BS and 6DS, named as Ta-B-S/TPK and Ta-D-S/TPK. were cloned. All of the three genes contain 6 extrons and 5 introns. and their ORF contain 1206 nucleotides corresponding to a putative 401aa protein. The fifth intron in Ta-B-S/TPK is longer than that of Ta-D-S/TPK and Hv-S/TPK. All of the variations in the sequence of protein are located in the C terminus and N terminus, which leading to the C terminus and N terminus of Hv-S/TPK fold toward inside. while C terminus and N terminus stretch out in Ta-B-S/TPK and Ta-D-S/TPK.To futher study the resistance mechanism of Hv-S/TPK. fungal development and H2O2 localization after Bgt inoculation in leaves of Yangmai158. T6VS·6AL and transgenic plants have been observed. In Yangmai158, fungi developed normally and the leaves were covered with hyphae and conidia chain s at 120 h:in the T6VS·6AL,there were two types of fungal development patterns:(1) Haustorium could not form and hypersensitive response was observed at 24 h. (2) The haustorium effectively formed in 15% of conidia at 24 h. however, the hyphae elongated more slowly than in the susceptible Yangmai158. At 120 h. the brown staining of the whole cell was detected beneath the secondary penetration peg. but the secondary hyphae and the colony did not form:in the transgenic plants, no haustorium could form and hypersensitive cell death was observed at 24 h in the Bgt attacked cells. The semi-quantitative RT-PCR indicated that in resistant H.villosa and T6VS·6AL Hv-S/TPK was upregulated by Bgt. in transgenic plants the expression was constitutive and high, but in Yangmai158 the expression of S/TPK was at a maintained at a relatively low level all the time. The resistance of Hv-S/TPK is involved with its expression level.A transient expression system was conducted in Yangmai158 to investigate the function of Hv-S/TPK and Ta-D-S/TPK Target genes were co-transformed with GUS gene into the epidermal cells of susceptible Yangmai158 by gene gun. After transformation, leaf surface was inoculated with powdery mildew spores. Forty-eight hours after inoculation, haustorial index was calculated in the GUS expression cells. The results implied that two gene, when transiently expressed in leaf epidermal cells of susceptible wheat variety, could disrease the haustorium index dramatically. However. Ta-D-S/TPK has no resistance function in susceptible Yangmai158, it may be that the promoter of Ta-D-S/TPK could not be activated by Bgt.Because Pm21 and Hv-S/TPK was located on the same chromosome region, to screen more resistance gene analogues in this resion, the RGAs surrouding the Hv-S/TPK was isolated using sequence of rice chromosome 2 as the reference.59 BACs on the rice chromosome 2 flanking Hv-S/TPK was screened for RGAs. which were then used as templates to search the homologous ESTs in the Graingene database for primer pairs designing. A pair of primers (14940F/14940R) was designed based on the sequence of Barley 1-14940. and used to screen the cDNA library of H. villosa. A full-length cDNA clone was obtianed. Sequence analysis showed that this full-length clone contains 2491 nucleotides and its putative protein contains a conserved NAF domain and a kinase domain. This clone was named as Hv-CIPK. which belongs in SnRK3 subfamily. Hv-CIPK was located in the short arm of 6V chromosome of H. villosa. Semi-quantitative RT-PCR showed that gene expression remains steady in H. villosa. Hv-CIPK gene was transformed into Yangmail58 and 68 positive transgenic plans were obtained.some of which showed high resistance to powdey mildew. |
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