Deoxynivalenol Toxin Of Fusarium Graminearum And Cloning And Transformation Of Toxicity Degradation Gene Tri101

Fusarium head blight (FHB), caused by Fusarium spp., is one of major disease of grain species. Fusarium graminearum Tri101 encodes a trichothecene 3-O-acetyltransferase that converts DON to a less toxic acetylated form. The main purpose of this paper is to research deoxynivalenol toxin of Fusarium graminearum and cloning, expression and transformation of toxicity degradation gene Tri101 in wheat, lay the theoretical basis for the detoxification of food crops and the effective control of the F. graminearum,and provide the scientific bases for searching for economy, safety and high efficiency purification method of mycotoxin in food and feed. The main results were summarized as follows:1. The basic morphology of Fusarium graminearum 0623 was observed by culturing it for 3d , 5d and 7d in PDA medium. Aerial mycelium grew more and more luxuriantly. The central old mycelium was sunk after 7d. With increasing day of culture, colonial stroma slowly changed from white to red, and a part of it was dark red. The spore morphology of it in greenbean soup for 3d was sickle shaped. Fusarium graminearum 0623 was cultured for 10d, 20d, 30d, 40d and 50d in wheat medium in order to detect DON content in different times with HPLC. Tests showed DON content was most highly when it was cultured for thirty days. However, DON content was fast setback after 30d.2. The cDNA of Tri101 was amplied from the Fusarium graminearum 0623 total RNA by RT-PCR. The results showed that Tri101 gene was 1356 bp in size and encoded 451 amino acids (GenBank accession number:GQ907236).The molecular weight (Mw) and theoretical isoelectric point (pI) is 49.45 kD and 5.14 respectively. The deduced amino acid of Fusarium graminearum 0623 Tri101 gene shared 99.56% homology with the deduced amino acid published by Kimura in GenBank. Alignments with the deduced amino acid of mature proteins in other 13 species of Fusarium showed 97.91%~75.68%. Based on Tri101 amino acid sequence the phylogenic tree of trichothecene 3-O-acetyltransferase gene was established. The results showed that Fg0623 and F. sporotrichioides belong to the same group of organisms; it was closely related to F. asiaticum while distantly related to F. oxysporum, F. moniliforme, F. nygamai, F. nisikadoi and F. decemcellulare.3. The Tri101 gene was ligated to the expression vector pGEX-4T2. The recombinant plasmid, pGEX-4T2/Tri101 was then transformed into E.coli BL21 strain, induced by IPTG. The results of SDS-PAGE and Western blot analysis showed that the Tri101 gene was highly expressed. The molecular weight of the recombinant fusion protein was 75.45 kDa, consistent with the predicted result. Polyclonal anti-GST-Tri101 rabbit antibody was successfully prepared by using purified Tri101 protein as immunogen. The ELISA titer of antiserum against GST-Tri101 was about 1∶2 56 000. Western blot analysis showed that the antiserum could specifically bind to the expressed Tri101 protein. Trichothecene 3-O-acetyltransferase encoded by Tri101 gene in Fusarium-damaged kernels of wheat was successfully detected with the antibody.4. The plant expression vector pCAMBIA1301-Tri101, which was structured successfully, was transformed into Agrobacterium tumefaciens EHA105 by liquid nitrogen cryopreservation. Shoot apical meristem cells of YuMai 18, ZhengMai 366 and ZhengMai 9962 of were transformed by pCAMBIA1301-Tri101-EHA105. After selected with solution containing 100mg/L hygromyci, the Tri101 gene transgenic seedlings were then proved by PCR. The result indicated that 32, 15 and 29 T0 plants were obtained. GUS staining identification showed that blue dots.It was proved that the Tri101 genes were integrated into the wheat genomes and they could express in the transgenic wheat.