| Wheat Fusarium head blight (FHB) or scab is a destructive disease in the warm and humid regions of the world. In China, Fusarium graminearum (sexual stage:Gibberella zeae) is the predominant pathogen responsible for the epidemic of wheat scab disease. FHB not only causes yield loss, but also decreases the grain quality, which brings the public concerns of food safety. The primary concern is the contamination of grain with fungus-produced trichothecenes toxins, which have been proved to be harmful to both human and animal health. Deoxynivalenol (DON) is one of the most important toxin members. Breeding of wheat cultivars with FHB resistance is considered to be the most economical, the safest and most effective strategy for the control of FHB. Intensive studies have been concentrated on the genetics analysis, QTL mapping of FHB resistance as well as physiological and biochemical mechanism of host resistance. However, the cloning of FHB resistance related genes and the understanding of molecular mechanism of FHB resistance is quite limited. The development and use of mutants is very important for functional genomics, especially for those complex resistance traits such as FHB resistance. In our previous study, a stable FHB susceptible mutant NAUH117 was identified by fast neutron irradiation of the dry seeds of Wangshuibai. It was further found that multiple complex structural rearrangements resulted the deletion of the major FHB resistance QTL on chromosome 3BS in NAUH117, and this led to significant increased susceptibility of the mutant. In the present research, by using the Affymetrix Wheat Genome GeneChip, the transcription profiles were analyzed and compared in the wild-type Wangshuibai and NAUH117 upon the inoculation of Fusarium graminearum and DON. Based on the results of expression analysis and in silico chromosome localization, a non-specific lipid transfer protein gene were further selected and cloned in Wangshuibai. The main results obtained are as following:1. Comparison of expression profiles of Wangshuibai and NAUH117 upon the inoculation with Fusarium graminearumGene expression in spikes of Wangshuibai and NAUH117 inoculated with Fusarium graminearum (Tester) and water (Control) was profiled with Affymetrix Wheat Genome GeneChip. Using the threshold value p<0.05 and fold>2, differentially expressed genes between the FHB-inoculated and the corresponding mock-inoculated samples for both genotypes were selected. A total of 2,981 genes were differentially expressed induced by Fusarium graminearum in Wangshuibai. Among them,1,874 genes were up-regulated and 1,107 genes were down-regulated. A total of 3,813 genes differentially expressed induced by Fusarium graminearum in NAUH117. Among them,2,143 genes were up-regulated and 1,670 genes were down-regulated. Using the Blast2go software, it was found that 1,523 up-regulated genes of Wangshuibai had function annotation information,753 up-regulated genes had GO assignments; 1,846 up-regulated genes of NAUH117 with function annotation information,1,041 up-regulated genes had GO assignments. The regulations of biological quality, anatomical structure development, multicellular organismal development, nucleoside binding, lyase activity were only found in NAUH117. KEGG pathway analysis showed that the up-regulated expressed genes in Wangshuibai and NAUH117 involved in the plant disease resistance signaling pathways and the plant-pathogen interaction. The plant disease resistance signaling pathways included plant secondary metabolism, active oxygen burst, calcium ion, phosphatidic acid molecular, salicylic acid, jasmonic acid and ethylene signaling pathway; and the plant-pathogen interaction related to PAMP-triggered immunity and Effector-triggered immunity reactions, suggested that these disease resistance signaling pathways involved in FHB resistance. In order to determine the relationship of the differentially expressed genes with those previously mapped QTLs, these up regulated genes related to defense response were chromosomal localized in silico. A total of 231 up-regulate genes (12.40%) of Wangshuibai and 197 up-regulate genes (9.20%) of NAUH117 were mapped.2. Comparison of expression profiles of Wangshuibai upon the inoculation with Fusarium graminearum and DONGene expression in spikes of Wangshuibai inoculated with DON (Tester) and water (Control) was profiled with Affymetrix Wheat Genome GeneChip. Using the threshold value p<0.05 and fold>2, differentially expressed genes between the DON-inoculated and the corresponding mock-inoculated samples were selected. A total of 1,114 genes were differentially expressed induced by DON, with 949 genes were up-regulated and 165 genes were down-regulated. Using the Blast2go softwares, it was found that 761 up-regulated genes had function annotation information,490 up-regulated genes had GO assignments. The transporter activity, oxidoreductase activity, cofactor binding, structural constituent of ribosome, ligase activity, peroxidase activity, enzyme inhibitor activity, hormone metabolic process, oxidation reduction, response to chemical stimulus, macromolecule localization, signaling pathway and cellular response to stimulus were only found in Wangshuibai in response to Fusarium graminearum inoculation. KEGG pathway analysis showed that the up-regulated expressed genes of Wangshuibai in reponse to Fusarium graminearum and DON inoculation involved in the plant disease resistance signaling pathways and the plant-pathogen interaction, suggesting that these disease resistance signaling pathways involved in resistances to FHB and DON. In order to determine the relationship of the differentially expressed genes with those previously mapped QTLs, these up regulated genes related to defense response were chromosomal localized in silico. A total of 110 up-regulate genes (11.60%) of Wangshuibai in response to DON inoculation were mapped.3. Cloning of a non-specific lipid transfer protein (LTP) gene in WangshuibaiAn up-regulated probe (EST-432), which was mapped to the 3BS was selected for cloning. A pair of primers were designed and used for PCR using the cDNA of Wangshuibai. Based on the sequence information, a full-length ORF of a LTP cDNA gene was cloned from Wangshuibai. Using Chinese Spring nulli-tetrasomic and deletion lines of homoeologous group 3, the gene was located to the region of FLO.78-0.87 on 3BS, which is the same region as the major QTL for FHB resistance, Fhb1. The gene was designated as TaLTP-B. Sequence analysis indicated that its ORF was 348 bp, coding a protein of 115 amino acids. The primary structure of the putative TaLTP-B protein contained a signal peptide, including a pattern of eight-cysteine in a conserved position. Subcellular localization assay showed that the fusion protein of TaLTP-B and the green fluorescence protein (GFP) were targeted to the cell wall. qRT-PCR analysis showed that, the transcript level of TaLTP-B was up-regulated in the spikes of Wangshuibai when inoculated with Fusarium graminearum and DON, while no product was detected as this gene is not present in NAUH117, which has a fragment deletion of 3BS. In the podery mildew resistant wheat relative species Haynaldia villosa, the expression of TaLTP-B could also be significantly induced rapidly in response to Bgt. These results indicated that the TaLTP-B play important roles in the defense responses to both Fusarium graminearum and Bgt. In addition, we found that the expression of TaLTP-B in the leaves of Wangshuibai was up-regulated by ET, H2O2 and JA, suggesting that TaLTP-B may be involved in defense responses to fungus pathogen via the ET, H2O2 and JA signaling pathway. In order to elucidate the function of TaLTP-B, an over expression vector of TaLTP-B was constructed and transformed into the callus of a moderate susceptible variety Yangmail58 by the Genegun bombardment method. Totally,70 regenerated plants were obtained, in which 11 were identified as the positive transgenic plants by PCR. The preliminary evaluation result of the plants at To generation showed that the over expression of the TaLTP-B could increase the FHB and powdery mildew resistance of Yangmai 158. |
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