| Haynaldia villosa Schur (2n=14, VV), whose synonymous name is Dasypyrum villosum Candargy, is a diploid relative of wheat. It is an important genetic resource for wheat disease resistance breeding. Especially, on its short arm of 6V chromosome, a major gene Pm21 with high powdery mildew resistance has been transferred to wheat in breeding programs successfully.To clone genes involved in resistance to powdery mildew and investigate resistance mechanism in H.villosa, cDNA from the resistant H.villosa and susceptible mutant both inoculated with Blumeria gramimis f.sp. tritici Marchal (ab. Bgf), a fungus who can cause powdery mildew, and cDNA from the uninoculated resistant H. villosa were hybridized to the Barley 1 microarrays respectively in our laboratory. And those genes that were significantly up-regulated expressed in inoculated resistant H.villosa or differently expressed between inoculated resistant and susceptible plant were identified. Based on information of these differential expressed genes, ten disease relative gene sequences corresponding to ten up-regulated probes were selected as templates for PCR primer pairs designation, and PCR amplification were performed in cDNA of H. villosa inoculated with Bgt. Six cDNA fragments were obtained out of ten. Among these cDNA fragments, the one amplified with primers designated from probe Contig3687 showed 84% identity with O.sativa (Japonica Group) EREBP transcription factor, and the one amplified with primers designated for Contig 5017 showed 90% identity with O.sativa(japonica cultivar group) pto kinase interactor 1. Then the two primer pairs were screened independently in cDNA library made from resistant H.villosa inoculated with Bgt, and clones containing inserts representing a full length Hv-EREBP (Ethylene responsive element binding protein, EREBP) (GenBank accession FJ711058) gene and a full length Hv-PKI (Protein kinase interactor, PKI) (GenBank accession FJ711059) gene were obtained respectively.Tne clone containing a full length Hv-EREBP gene has a 1429bp insert, in which an Hv-EREBP gene with a 1185bp complete ORF was identified. Composed of 394 amino acids, the deduced protein of this Hv-EREBP gene encoded a complete AP2 domain. This protein has a 66% identity with O.sativa transcription factor EREBP1 (GenBank accession BAD 19536). Subcellular localization analysis showed that EREBP-GFP fusion proteins expressed intensively in the cell nucleus of onin epidermal cells, and the CDS fragment from the start code to the AP2 coserved domain encoded the amino adid sequence required for accurate positioning of this protein. Expression of the fusion protein Hv-EREBP was carried out with the pET expression system successfully, a powerful system developed for the cloning and expression of recombinant proteins in protease deficienct bacterial strains, i.e. E. coli. BL21(DE3). Recombinant plasmid was constructed by insertion of the full length Hv-EREBP gene into the pET32a vector, and transformed into the competent host cell BL21(DE3). After induction with the addition of IPTG to the bacterial culture, compared with the controls, the host stains contained the recombinant plasmid has yielded a specific band whose molecular weight was equal to the deduced theoretical molecular weight of the fusion protein Hv-EREBP, i.e. about 66.8KD. Real-time RT-PCR analysis showed that transcription of the Hv-EREBP gene was up-regulated significantly in H.villosa after inoculation with Bgt or application with exogenous ethylene, the gene transcription was suppressed in H.villosa treated with exogenous salicylic acid, and transcripts of this gene decreased gradually in H. villosa sprayed with exogenous jasmonic acid. Deduced from above observation, the Hv-EREBP gene must play a role in powdery mildew resistant pathway or ethylene defense pathway in H.villosa.Tne clone containing a full length Hv-PKI gene has a 1519bp insert, in which a 1089bp complete ORF representing a full length Hv-PKI gene was identified. The Hv-PKI gene encoded a deduced protein composed with 362 amino acids, in which a complete protein kinase domain was identified. This deduced protein has 93% identity with O.sativa protein kinase interactor (GenBank accession AAS98413). Subcellular localization analysis showed that PKI-GFP fusion proteins expressed intensively in the cytoplasm and on the membrane of onin epidermal cells, the CDS fragment from the start code to the coserved domain encoded the amino adid sequence required for accurate positioning of this protein. Expression of the fusion protein Hv-PKI was carried out with the pET expression system successfully. Recombinant plasmid was constructed by insertion of the full length Hv-PKI gene into the pET32a vector, and transformed into the competent host cell BL21(DE3). After induction by IPTG, compared with the controls, the host stains carrying the recombinant plasmid has produced a unique band whose molecular weight was equal to the deduced molecular weight of Hv-PKI protein, i.e.63.3KD. Analyzed by Real-time RT-PCR, expression of this gene was induced remarkably by exogenous hormone salicylic acid or Bgt. On the contrary, no sharp expression increase or decrease was detected in leaves of H.villosa after application with exogenous ethylene or jasmonic acid. Deduced from above observation, this gene must involve in powdery mildew reisitance and salicylic acid defense pathway in H.villosa.To clone candidate gene for Pm21, suppression subtractive hybridization was conducted between an isogenic resistant line carrying Pm21 and its recurrent parent Yangmai 5 by Chen et al., and a resistance gene analog (RGA) Ta-LRR2 (GeneBank accession ABQ53156.1) co-segregating with Pm21 was obtained. Afterwards, a resistance gene analog polymorphic molecular marker CINAU16 (NAU/XiBao 16) linked to Pm21 was developed from Ta-LRR2 sequence. Ta-LRR2 had an increased expression level in the resistant wheat T6VS/6AL after inoculation with Bgt. In this work, a homologous RGA sequence Hv-LRR encoding 226 amino acids (GenBank accession FJ711057) was cloned, assigned on the short arm of 6V chromosome in H. villosa. Expression of the Hv-LRR was dramatically induced in leaves of H.villosa challenged by Bgt, but not induced by exogenous hormones, i.e. ethylene, salicylic acid, jasmonic acid. This gene was deduced taking part in the powdery mildew resistant pathway in H. villosa.From our experience, tissue culture system for H.villosa was still quite difficult to be established at present time. To fast evaluate gene function, an effective and persistent virus-induced gene silencing (VIGS) system was established with barley stripe mosaic virus (BSMV) for H.villosa. Fluorescence observation of GFP gene expression showed that BSMV:GFP can deliver systemically from leaf to leaf in the early growing stage of the plant after inoculation. Characterization of phytoene desaturase (PDS) gene silencing process and Real-time RT-PCR detection showed that BSMV:PDS carrying reverse inserted fragment of PDS gene can infect H.villosa systemically, and suppress PDS transcripts as early as six days after inoculation. The photobleaching phenotype occurred mainly on the newly upper leaves, and PDS gene silencing process carried thorough the whole growing period in H.villosa. This was the first report that BSMV was used successfully for VIGS in a wild relative species of wheat. The established VIGS system will surely be a powerful reverse genetics tool for gene function study in H.villosa. With the established VIGS system, function analysis of two genes, i.e. the Hv-EREBP gene and a serine/threonine protein kinase(Hv-S/TPK) gene, were carried out. Cloned by Cao et al. in our laboratory previously, the Hv-S/TPK gene was a candidate gene for Pm21, and located on the short arm of 6 V chromosome in H. villosa. Expression of the Hv-EREBP gene or the Hv-S/TPK gene was dramatically down-regulated in plants inoculated with BSMV:Hv-EREBP or BSMV:Hv-S/TPK. Infection percentages by Bgt increased on leaves of H. villosa in which Hv-EREBP or Hv-S/TPK were silenced, and resistance to powdery mildew decreased. All these results showed that Hv-EREBP and Hv-S/TPK definitely played a role in powdery mildew resistant pathway in H.villosa. |
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