The Characterization Of DON Induced Genes And Promoters In Wheat

Fusarium head blight (FHB) is a fungal-type disease in triticeae crops which not only has negative effects on the quality of wheat but also cause a great loss in warm and humid regions or years. With the development of molecular biology, transgenic technology has a wide application prospects in the exploration of the interaction on wheat and pathogens and the wheat quality improvement or modification. The pathogenesis mechanism is not fully understanding and the shortage of valuable resistance genes have become the main bottlestem of transgenic technology in wheat FHB resistance breeding. Consequently, researches on the interaction of wheat and Fusarium graminearum (FG) and identification of fungal resistance related genes in wheat is a possible way to understand the mechanism of wheat FHB which contributes to the molecular wheat breeding.There are 6 genes that up regulated by DON were cloned from wheat cultivar Zhengmai 9023 by rapid amplification of cDNA ends. These genes are named An, Un, C7, 2100,221 and 2140 respectively. The results of bioinformatic analysis and multi-sequence alignment for amino acid showed that the functions of An and Un genes are unknow, C7 encodes the methionyl tRNA synthetase,2100 is a cysteinyl tRNA synthetase gene, the translation production of 221 gene is multidrug and toxin compound extrusion protein and the gene of 2140 encodes 1,3-β-glucan synthase component family protein. The expression vectors for subcellular localization were constructed by fused the 6 target genes with gfp. At the same time, the over expression vectors of these genes were constructed used for Agrobacterium mediated transformation.The target genes fused with gfp was used for subcellular localization of onion epidermal cell transient transformation by microprojectiole bombardment, it showed that 221 encodes a membrane protein in higher plants, the proteins translated by C7 and 2140 were in the nucleus, the fluorescence signal of 2100-gfp was in the cytoplasm or organelles, however the proteins encode by An and Un genes were localized in both membrane and nucleus.Genetic transformation and over-expression of the 6 candidate genes in Arabidopsis thaliana was mediated by Agrobacterium. An spray inoculation identification and DON tolerance tests were carried out in T2 generation transformed plants, the results showed that the transgenic plants of C7,221, Un and 2140 performed a significant tolerance against both DON and Fusarium conidia, meanwhile the tolerance of An and were improved, but no significant difference compared with wild type, and the resistance of 2100 was just the same as wild type control. The mycotoxin quantitative analysis of inoculated plant material was measured by liquid mass spectrometry, it showed that the DON content of all the transgenic lines were lower than wild type control except for 2100.The inducible promoters An and Un induced by DON were cloned from Zhengmai 9023 by TAIL-PCR. The expression vector of the 5’-deletion promoter fragment was constructed by inserted the gus gene as report gene. The genetic transformation of wheat was medidated by Agrobacterium GV3101. According to the PCR assay, there are 12 PCR positive lines in TO generation. In detail, WAnPl are 7, WAnP2 have only one, WUnPl have 2 and non positive line was identified of WUnP2. Transient expression of gus in immature embryos callus and stable expression of transgenic lines indicated that these two promoters have a normal function in gene transcription. The semiquantitative RT-PCR analysis confirmed that both An and Un promoters are DON inducible promoters in higher plant, the expression efficiency is regulated by DON.