| Fusarium head blight (FHB) caused by Fusarium graminearum is one of the destructive diseases of cereal crops including wheat, barley and maize. FHB not only causes yield loss and deteriorating seed quality, but also causes food contamination with trichothecenes toxins, which are harmful to both human and animal health, thus lead to the serious food safety issues. DON is one of the virulence factors and plays an important role in fungal pathogenesis. The improvement of FHB resistance by the development of disease-resistant cultivar is the most economical and effective way to control FHB and grain DON content in wheat.Based on the expression analysis of DON-induced Wangshuibai that confers high resistance of FHB by using Affymetrix microarray, a UDP-glycosyltransferase gene was found to be up-regulated by DON inoculation and the gene full length, named as TaUGT3, was cloned by Ma et al (2009). Transformation of TaUGT3 into Arabidopsis c.v. Columbia could enhance its tolerance against DON. Pei et al (2011) obtained the transgenic wheat by the bombardment method into wheat variety Alondra's. He further found that the transgenic lines of T1 generation showed enhanced FHB-resistance.Based on the comparison the differentially expressed genes induced by Fusarium graminearum in the spikes of Wangshuibai and the FHB-susceptible mutant NAUH117 using Affymetrix microarray, Zhao et al (2011) found that proline rich protein genes were down-regulated in the mutant NAUH117 and three members of PRP genes located in three wheat homoeologous group 3 chromosomes, namely TaPRPl, TaPRP2 and TaPRP3, were cloned. One of them, Ta-PRP3 located in the 3D chromosome was transformed into common wheat variety Yangmail58 and the transgenic plants of To generation showed enhanced FHB-resistance.In the present study, the TaUGT3 T2 and T3 transgenic lines in the background of Alondra's and the Ta-PRP3 Ti transgenic lines in the background of Yangmai158 were further characterized by PCR, qPCR to confirm the presence of the target gene and their expression pattern. The identified transgenic lines were further evaluated for their FHB resistance and agronomic traits to illustrate their effects on FHB-resistance and other charaters. The other PRP gene Ta-PRP1 located in the 3BS, in which the Fhbl locus was located, was transformed into the Yangmai158 in order to figure out its function in FHB resistance. The main results are as follows:1. Characterization of the function of TaUGT3 in FHB-resistance and DON accumulation of wheatSpecific joint primer pairs covering the promoter and the target gene TaUGT3 sequences were designed and used for PCR to identify the positive plants of the TaUGT3 transgenic lines at both T2 and T3 generation. Five transgenic lines were identified to be positive. Part of the plants from four T2 lines was chosen to perform qRT-PCR to analyze the expression pattern of the transgene in the spikes. The results showed that the target genes were up-regulated in T2-31 and T2-92 compared with the receptor control Alondra's. FHB-resistance evaluation using single floret inoculation method of the 5 transgenic lines showed that,12 days after inoculation, the proportion of scabby spikelets showed significant differences between the transgenic lines T2-31, T2-92 and Alondra's. While 18 days after inoculation, the differences were not obvious between the two lines and the control. The proportion of scabby spikelets of line T2-26 was significantly different with thecontrol only at 16 days after inoculation, while there were no significant differences at 10 and 21 days after inoculation. For all the time point, the T2-20 and T2-54 showed no significant differencewith the control. These results indicated that TaUGT3 might have some extend effects on the delay of the spreading of the Fusarium pathogen at an earlier period. Part of the plants from the 5 lines was chosen to determine the grain DON contents using Enzyme-link Immunosorbent Assay (ELISA) kit. The results showed that the DON contents of T2-26 and T2-92 were significantly lower than that of the control, indicating that TaUGT3 had some extend effects on the detoxification of grain DON. The agronomical character investigation of T2 and T3 transgenic lines showed that the plant heights of T2-20, T2-26 and T2-92 were significantly shorter than those of the control, the T3-26 and T3-31 had more tillers than that of Alondra's, and the panicle length and the number of spikelets of T3-31 showed significant difference compared with those of Alondra's.2. Characterization of the function of Ta-PRP3 in FHB-resistance of wheatAccording to the sequence of the vector for transformation, the specific joint primer pairs covering the 35S promoter and the Ta-PRP3 gene sequences were designed and used for identification of the T1 and T2 transgenic plants by PCR. The results showed that 7 of the T1 lines and 5 of the T2 lines could amplify the target fragment of 550bp and were identified to be positive transgenic lines. Part of the T1 plants from 6 lines was selected for qRT-PCR and it was found that that the target genes in some plants from T1-2, T1-9 and T1-15 were up-regulated compared with Yangmai158. The evaluation of FHB resistance of the 7 positive T1 transgenic lines using single floret inoculation method showed that the average proportion of scabby spikelets of lines T1-2, T1-9, T1-18, Ti-115, Tr12 and Ti-14 were significantly different compared with the control Yangmai 158, indicating that the Ta-PRP3 plays an important role in resistance to FHB in wheat. Agronomic traits investigation of T1 positive lines showed that T1-9 had a significant difference in average number of tillers, compared with Yangmai 158; T1-12 and T1-15 were significantly shorter than Yangmai 158; T1-18 was significantly taller than Yangmai 158. The agronomical character investigation of T2 transgenic lines showed that T2-14 had more tiller number and it was taller than Yangmai 158; the panicle length of T2-2 and T2-12 were significant higher than Yangmai 158; the number of spikelets of T2-3 showed significant differences compared with Yangmai 158.3. The transformation of Ta-PRP1 into wheat variety Yangmai 158To characterize the function of Ta-PRP1 in FHB resistance of wheat, an over-expression vector pBI-Ta-PRPl-HA was constructed and co-transformed into callus of the moderate FHB susceptible wheat variety Yangmai 158 by particle bombardment together with the vector pAHC20, which carrying the Bar gene. Totally,31 To regenerated plants were obtained,8 of which were identified as positive transgenic plants by PCR analysis. |
PDF Full Text Request