Construction Of A Triple Antifungal Fusion Gene And Identification Of Resistance To Fungi Of Transgenic Tobacco Plants

Plant protection is a major challenge to agriculture worldwide.Significant yield losses due to fungal attack limits crops productivity and canbe very severe local epidemic infections. Transferring antifungal protein genesto plants becomes a most direct and efficient way to obtain fungi resistantplants and has attracted many researchers' attention because of its highbio-safety and long duration. With more and more antifungal proteins werefound and applied, the problem of multi-gene transforming and expressionarose. Gene fusion, as a novel technology, is a most efficient way to solve thisproblem. In this study, we constructed a fusion gene with tobacco class Ichitinase gene, wheat thionin gene and tobacco class I β-1,3-glucanase gene,and transferred corresponding expression vectors to tobacco, then realized theco-expression of the three antifungal protein genes and enhanced resistanceagainst fungal disease. The main results are as follows: 1. Three antifungal protein genes, tobacco class I chitinase gene, wheatthionin gene and tobacco class I β-1,3-glucanase gene were cloned andmodified slightly used to construct two fusion genes, FG1, FG2, and FG2contains a short carboxyl-terminal propeptide that mediates transport to plantvacuole. 2. Three plant expression vectors containing the fusion genes wereconstructed, and named pBI-FG1, pBI-FG2 and pMR-FG1, respectively. Inaddition, another three vectors containing CHI, THI, GLU, respectively, wereconstructed as control. 3. Six group transgenic tobacco plants were obtained by the standard leaf —3—张岳民:三价抗真菌融合基因植物表达载体的构建及转基因烟草的抗病性分析disc transformation method using kanamycin selection. 4. Expression analysis of transgenic tobacco plants containing FG1 orFG2 constructs showed that the FG1 was secreted extracellularly, while FG2was sorted to the vacuole because it has a targeting information resides in thecarboxyl-terminal propeptide. In addition, they retained their biologicalactivities respectively. 5. The performance of tobacco plants expressing the fusion gene in vitroand in vivo assay revealed significantly enhanced protection against fungalattack when compared with control plants. 6. Northern and Western blot analysis in pMR-FG1 and pBI-FG1transformants showed that pMR-FG1 can improve the gene expression both intranscription level and in translation level.