| Wheat powdery mildew, caused by Blumeria graminis(DC.) Speer f. sp. Tritici (Bgt), is fungal disease occurred all over the world. The distribution of powdery mildew was enlarging because of the improved irrigation and fertilization, moreover, it was more severely in the moist areas. The wheat could be infected by Bgt from one-leaf phase to old. Tillers of wheat decreased when it was infected in the early development phase, and the number of seed reduced when the leaves and ear were infected. Powdery mildew can be controlled to a certain extent by changing tillage pattern which was usually limited by climate and so on.Effectively chemical methods usually increase the inputs including manpower, material and financial resources, and result in severe environmental pollution and damage to our healthy for a long time. It was a very effective and economical method for controlling the disease to plant resistance cultivars. But the resistance was easily lost because of the variation of Bgt physiological race. Durable resistance cultivars with many Pm genes cultivated by traditional or gene-engineering methods was major researched purpose, while, the study of resistance mechanism was the foundational work to achieve the aim.Wheat landrace Hongyoumai showed good resistance to Btg preserved by the lab and displayed immune. F3 homogeneous resistant and susceptible generations from the cross of Hongyoumai×Yumail3 (respectively, represented resistant and susceptible pools) were used to hybrid with affymetrix gene chip and two treatments were set pre-induced control and 24 hours after inoculation. A lot of transcripts were obtained through comparisons of genechip data. The probes of genechip were primarily analyzed by in silicon cloning and real time PCR in this study, and results as follows:1. Cloning of resistance-related genes in WheatProbes of the resistance-related genes were selected according to the results of genechip. The primers were designed on the basis of the full-length DNA sequences of genes obtained by in silico cloning. Leucine-rich repeat protein, receptor-like kinase protein, serine/threonine kinase, pathogenesis-related protein 1, pathogenesis-related protein 2 and three pSBGer proteins were successfully cloned in susceptible cultivar Yumail3 and resistant Hongyoumai at the same time. Mlo was also cloned in Yumail3 and Hongyoumai, sequences analysis showed that just only one nucleotide acid was different between two DNA sequences and 90% idential with Mlo from barley. Amino acid (aa) analysis indicated that original 18 aa were signal peptide and contained 7 trans-membrane domains. The aa during 414～435 was active site and 451,459,462,475 and 483 aa were glycosylated. Three-domensional structure predicted in the next analysis showed that the aa of 446 site is Ser in Hongyoumai and Gly in Yumail3, because of the difference of one nucleotide acid between these cultivars. Pm3HYM, one resistance gene homologous to Pm3b, was only cloned in Hongyoumai. The 4208bp of DNA sequence was of 3777bp ORF and encoded 1258 aa. Amino acid structure analysis showed that it contained P-loop NTPase and LRR domain. Pm3HYM maybe have special function because it was particular status by cluster analysis in all homological sequences. Above obtained genes were related to resistance reaction in plant. These results will lay foundation for the next analysis.2. Expression analysis by real-time PCRFive genes:pathogenesis-related protein 1.2(PR1.2), receptor-like kinase protein (RLK), serine/threonine kinase (STK), homological Mlo in Barley and Pm3HYM were analyzed by real-time PCR for the gene expression pattern. The primers were designed according to the sequences of genes, and the expression analysis induced by Bgt during pathogenesis (72 h or 96 h) was analyzed by real-time PCR. The expression peak of PR1.2 in HYM appeared in the time-point of 18 h, while,72 h in YU13. The expression peak of RLK was 24 h in YU13, but not obvious peak in HYM. The expression peak of STK in HYM appeared in the time-point of 18 h, but the rising current in YU13 was invisible. The Mlo in YU13 highly expressed in 72 h, while, the expression peak appeared 12 h in HYM. Pm3HYM was induced and ascended right now in Hongyoumai, and the peak appeared in 12 h. Except for gene PR1.2 and RLK, the expression patterns of the genes were concordant with genechip. It was showed that the expression patterns of genes and the time peak of expression was directly related to resistant reaction. 3. Construction of VIGS vector Pm3HYMIn order to confirm the function of cloned genes in the resistance reaction, the target gene need be silent in silencing system in order to validate the gene function that is effectively induced by Barley Stripe Mosaic Virus in monocotyledon. Pm3HYM was the target gene in this experiment. Two DNA fragments silenced zone were cloned from the pMD19-T vector connected to Pm3HYM genes by PCR. Then silencing fragments were linked to y vector by incision enzyme and ligase. The construction of VIGS vector was correct by PCR and double digests. The purpose of the experiment was to explore the process of VIGS construction and paved the way to the further study.
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