|
In agricultural production, plant diseases is a very difficult issue, especially fungal diseases, plant more than 80% of total disease are caused by fungi. Control of plant fungal diseases traditional methods are: (1) obtained by conventional breeding methods resistant varieties; (2) use of chemical pesticides; (3) take the crop rotation cultivation tillage. But practice shows that these three methods have certain drawbacks and limitations, can not fundamentally solve the problem of fungal diseases. In recent years, molecular biology and genetic engineering technology is developing rapidly, through recombinant DNA technology into anti-fungal genes and, ultimately, anti-fungal new varieties of plants, from the fundamental solution to the problem of plant fungal diseases. Poly-galacturonic acid inhibiting protein (polygalacturonase-inhibiting proteins, PGIPs) gene is more of a domestic and international research antifungal genes, PGIPs is an autoimmune associated with plants multifunctional protein, are plant extracellular leucine-rich repeats (extracellular leucine-rich repeat, eLRR) superfamily. Poly-galacturonic acid enzymes (polygalacturonases, PGs) are infected plant pathogenic fungi when the first hydrolytic enzymes secreted, it can hydrolyze the pathogens into the plant cell walls, plant cells, and secreted by the fungus PGIPs PGs with specific binding, by reducing the activity of PGs and accumulate in the plant defense responses to activate various long-chain oligomers of galacturonic acid (oligogalacturonides, OGs), ultimately to inhibit pathogenic fungi plant purposes. Torches Pear (Pyrus pyrifolia Nakai) Yunnan local pear varieties, soil adaptability, resistance to scab, canker on pear psylla also has a strong resistance, resistance to frost, cold tolerance is also strong, so the species The gene has a certain resilience research value. This study first cloned from torches pear PpPGIP gene and the gene for a series of full-length cDNA sequence bioinformatics analysis, found that the gene sequences with other species PGIP highly homologous; This gene encodes a protein sequence with multiple conserved LRR domain; its encoded protein three-dimensional structure of PvPGIP2 highly consistent with the bean, so make sure it is a torch pear PGIP gene. While using RT-PCR analysis PpPGIP torch pear leaves in different developmental stages and peel the expression level of transcription, the results show that the gene expression in the leaves is very low, but its expression in higher peel. Using molecular biology techniques to build PpPGIP constitutive plant expression vector pCAMBIA2300S-PpPGIP, and through the freeze-thaw method CaCl2 plant expression vector into Agrobacterium strain LBA4404 and EHA105 in. During the gene genetic transformation experiments before GUS staining experiments on tobacco through, carnations, lisianthus three receptors plant genetic transformation system optimization, through kanamycin resistance experimentally determined two kinds of flowers, carnations and lisianthus concentration of antibiotic screening plants. Agrobacterium-mediated genetic transformation method PpPGIP receptor gene into three kinds of plants, obtained through kanamycin transgenic tobacco 52, 13 carnations, lisianthus 65. By PCR analysis of genomic DNA obtained transgenic plants, including tobacco 24, lisianthus 8. RT-PCR analysis showed PPPGIP in 10 transgenic tobacco plant has been expressed. To further study the PpPGIP antifungal activity, in order to have obtained 10 PpPGIP expression at the transcriptional level of transgenic tobacco as a material, through inhibition experiments to evaluate PpPGIP flat in vitro antifungal activity. Experimental results show that, compared to wild-type tobacco, PPPGIP transgenic tobacco chosen for this experiment, 11 kinds of the five kinds of fungi have different degrees of inhibition, the five species of fungi were: fungi of the genus Phomopsis (Phomopsis sp. ), Piron mildew fungi (Peyroneuaea), Alternaria (Alternaria sp.), Aspergillus niger (Aspergillus niger) and Penicillium (Penicillium sp.).
|