Cloning And Identification Of Wheat Resistance-Related Genes And Cis-Elements Against Fusarium Head Blight

Fusarium head blight (FHB) of Tritium aestivum caused by Fusarium graminearum is a devastating disease around the worldwide, especially in warm and humid regions. It not only leads to yield loss, but also produces toxin resulting in a poor quality.Trichothecenes are kinds of mycotoxins produced by Fusarium graminearum and one of its members, trichothecene deoxynivalenol (DON) is the most common type. The results show that DON plays a decisive role in disease development and Fusarium graminearum expansion. Deoxynivalenol-nonproducing Fusarium graminearum casuses initial infection, but does not cause disease spread in the host tissue. In this study, we cloned and analyzed wheat genes and cis-elements induced by DON. Those are well suited for FHB resisitance research research and transgenic wheat research in general.The main results obtained are as follows:1. According to the published sequence of cyptochrome P450 CYP709C1, we cloned seventy-two coding sequences with Sumai 3 cDNA library. Sequence analysis showed that:there is no sequence the same with CYP709C1 completely, and only two of eight sequences are identical, indicating the big deal copies in Sumai 3. The full-length cDNA of CYP709C1 is 1545 bp in sevety-one clones, that means the highly conservative in phylogenesis. In addition, there is a sequence of 1553 bp containing a terminator in the middle, showing that can not code a complete protein. Meanwhile, we cloned five gDNA sequences with Sumai 3 gDNA. The results show CYP709C1 contain at least three intrrons. The introns length is not exactly the same in these five clones, mainly for the change in base, but splice site between intron and exon are identical. In addition, we cloned two CYP709C1 promoters. The 500 bp region close to the start. codon contain many important cis-elements after bioinformatics analysis, suggesting CYP709C1 plays an important role in response to disease resistance. In order to analyze the characteristics of its expression, the two promoters had been constructed into expression vector with the reporter gene-GUS, and then they were transformed into wheat calli by biolistic.2. According to the Zheng 9023 suppressive subtractive hybridization (SSH) cDNA laboratory, we isolated many EST sequences induced by DON. We cloned four full-length genes through rapid amplification of cDNA ends (RACE):Methionyl-tRNA synthetase、Cysteinyl-tRNA synthetase、MATE efflux family protein、1,3-β-glucan synthase. We also cloned the full-length gDNA of these four genes, and then analysed the genes structure. The genes copies were identified by Southern hybridization and the integrity of cloning was confirmed by Northern hybridization. We predicted the possible protein structure and targeting by bioinformatics. By constructing GFP fusion protein, transient expression in onion epidermal cells to analyze the subcellular localization.3. Wheat cultivars Zheng 9023 and Sumai 3 were treated by DON、Fusarium asiaticum 5035 and a mutant strain Tri5- respectively. Drawn at different times after inoculation, extracted RNA and reverse transcription. The results of Quantitative realtime-PCR show that these four genes are different expression in different periods. (1) In Zheng 9023, the expression of Methionyl-tRNA synthetase is correlated with DON accumulation, but not related to the Fusarium infection significantly; while in Sumai 3, the expression of Methionyl-tRNA synthetase gene are correlated with DON accumulation and Fusarium infection. (2) In Zheng 9023, the expression of Cysteinyl-tRNA synthetase gene is associated with toxin accumulation, and the expression is also high after the initial infection; while in Sumai 3 has the same results, but the expression is lower than in Zheng 9023 after the initial infection. (3) MATE efflux family protein expression is consistednt with DON accumulation and infection time in Zheng 9023, and there is the same result in Sumai 3. (4) In Zheng 9023,1,3-β-glucan synthase expression is coincident with the accumulation of DON, has nothing to do with the pathogen infection. Meanwhile,1,3-β-glucan synthase is induced by DON while inhibited by infection.