| Fusarium head blight (FHB) or scab is an important disease of wheat in warm and humid regions which causes a serious threat to grain yield, quality and food safety. Fusarium graminearum (sexual stage:Gibberella zeae) is the dominant pathogen responsible for wheat scab epidemics in China. Selection of wheat cultivars resistant to FHB is the most economical and effective means for FHB control. Since 1950s, intensive studies have been concentrated on pathogenicity and mechanisms of pathogenesis of Fusarium pathogen, screening of resistant resources, evaluation methods for host resistance, genetics analysis, QTL mapping and physiological and biochemistry mechanism of host resistance as well as improvement of disease resistance in wheat breeding. However, research on genes cloning related to scab resistance and molecular mechanism of FHB resistance is quite limited. Creation of mutants was of great value in the functional genomic research. Inducing and identification of mutants susceptible to FHB from resistant resources will be of great help for the clarification of mechanism of such a complex resistance trait at the molecular level. In the present investigation, several mutants susceptible to FHB from both Sumai3 and Wangshuibai were obtained by using fast neutron irradiation. A mutant designated as NAUH117 was characterized to have a deletion on chromosome 3BS which lead to its susceptibility. By comparing the genes differentially expressed in the wild-type Wangshuibai and its NAUH117 both induced by F. graminearum, molecular mechanism for FHB resistance was compiled and a receptor-like protein kinase gene was cloned. The main results are as follow:1. Inducing and identification of FHB susceptible mutants of Sumai3 and WangshuibaiWheat varieties Sumai3 and Wangshuibai have been accepted as the most important genetic sources for FHB resistance, and their resistances were complex quantitative traits controlled by multiple nucleus genes. However, major QTLs for wheat FHB resistance have repeatedly located on chromosome 3BS in both varieties. In this study,8 mutants susceptible to FHB from both Sumai3 and Wangshuibai were obtained by using fast neutron irradiation. Molecular marker analysis indicated that there were deletions on 3BS in 4 mutants, there was deletion of TaPDR1 on 5DS in 1 mutant, while the mutagenized loci of the remained 3 mutants need to be further identified.2. Morphological, molecular marker and inheritance analysis of the susceptible mutant NAUH117 from Wangshuibai1) Comparison of infection progress of pathogen in the inoculated spikes of Wangshuibai and NAUH117:Transformation of gfp gene to Fusarium gramiearum facilitated the observation of infection progress of pathogen in wheat spike by tracking the GFP signals under a fluorescence microscopy. Our results showed that, in Wangshuibai, Fusarium pathogen colonized only in the inoculated spikelet and stopped at the base of rachilla, while in NAUH117, Fusarium pathogen could permeate through the base of rachilla into rachis and infect the adjacent spikelets.2) Molecular marker analysis of the mutagenized locus for NAUH117:Eighty-six markers previously located on 3BS and 796 none-3BS markers were utilized for amplification in Wangshuibai and NAUH117. The results indicated that all the none-3BS markers showed no amplification difference between the two materials, while only some of the markers located on 3BS showed amplification difference. All the markers distributed on the 3BS deletion bins of FLO.57-1.00 failed to amplify the 3B specific bands in NAUH117. Among the four markers we used located in the 3BS deletion bin FL 0.56-0.57, two could not amplify the 3B specific bands while the other two could. Markers distributed on the 3BS deletion bins of FLO.33-0.56 could amplify the 3B specific bands. From these results, it is concluded that in NAUH117, there is a deletion on 3BS, and the breakpoint stays in the region of 3BS FL 0.56-0.57. A major QTL confering FHB resistance on 3BS (syn. Qfhs.ndsu-3BS, designated as Fhbl) was reported in the deletion bin 3BS 0.78-0.87. It is deduced that the Fhb1 was absent in NAUH117, and this leads to its increased susceptibility to Fusarium Head Blight.3) Genetic analysis of the mutated susceptible locus in NAUH117:The forward and reverse F1 hybrid, F2 and F2:3 populations derived from the cross between Wangshuibai and NAUH117 were evaluated for type II FHB resistance to study the susceptibility inherence of NAUH117. Comparison of the mean disease severity among Wangshuibai, NAUH117 and the F1 hybrid showed that PSS of F1 hybrids were similar as the wild-type Wangshuibai. Among a total of 436 F2 individuals or its derived F2:3 families,102 were susceptible to wheat scab. By x2 test, the segregation ration of resistant and susceptible fit 3:1, suggesting that the susceptibility of NAUH117 was controlled by single recessive gene. Further linkage analysis of the molecular markers and the resistance phenotype of the F2 individuals confirmed the deletion on 3BS in NAUH117 should be the major reason of its increased FHB susceptibility.3. Gene expression comparison of Wangshuibai and NAUH117 induced by Fusarium graminearum infection using the Affymetrix Wheat Genome GeneChipGene expression in spikes of Wangshuibai and NAUH117 inoculated with F.graminearum and water was profiled with Affymetrix Wheat Genome GeneChip. Differentially expressed probes (including up-and down-regulated beyond 2-fold) between the FHB-inoculated and the corresponding mock-inoculated samples for each genotype were selected. Seven up-regulated probes were selected and primers were designed to perform RT-PCR, and the results confirmed the credibility of the genechip results. Gene expression comparison of Wangshuibai and NAUH117 showed that some genes showed similar expression pattern both in the resistant wildtype and susceptible mutant. However, some were uniquely up-or down-regulated in Wangshuibai or NAUH117. Among those commonly up-or down-regulated genes, typically involving the Jasmonic acid(JA) pathway, Ethylene pathway(ET) pathway, the Phenylpropanoid pathway (PAL) etc. Those genes uniquely up-or down-regulated in wangshuibai included signal transduction related genes, cell senescence and apoptosis related genes, pathogenesis and denfence-response related genes, transporter and transferase genes, etc.4. Cloning of a receptor like kinase (RLK) gene near Fhbl from WangshuibaiCombining the results of Genechip assay and sequence comparative analysis of the collinearity region of wheat 3BS and Brachypodium distachyon, a full-length RLK cDNA gene, named TaNRLK-B, was cloned from Wangshuibai. Sequence analysis indicated that its ORF was 1140bp, coding a protein of 380 amino acids. SMART software predicted that the gene contained a signal sequence, an extra-cellular domain of unknown function, a trans-membrane region and an intracellular serine-threonine kinase domain. Using Chinese Spring nulli-tetrasomic and deletion lines of homoeologous group 3, TaNRLK-B was located on 3BS at the region of FLO.78-0.87, where the Fhbl was located. Both semquantitative RT-PCR and quantitative PCR analysis showed TaNRLK-B was up-regulated in the spikes of Wangshuibai when inoculated with Fusarium. No product of RT-PCR amplification was detected as it is deleted in NAUH117. An over expression vector and a RNAi vector of TaNRLK-B has been constructed, and the transformed into wheat by Agrobacterium tumefacious mediated shoot tip culture method and Genegun bombardment method. The regeneration of the transgenic plants and the identification of transformant were in progress. |
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