Study On The Transformation Of CHI Gene From Balsampear And AFP Gene From Motherwort Herb On Maize(Zeamays)

Maize is the primary raw material crop for food,·feed, medicine and chemical industry in modern society. In 2006, the planting acreage is about 2.69 million hectares, which accounts for 20% of total plough area in our countory. The fungal diseases is the major factor influencing the quantity and quality of corn, which reduce 10-20% of total yield every year. Fungal cure druggeries are mostly chemical pesticide.It would produce fungal drug resistance and contaminate environment. So it is the most economical method that select and breeding the disease-resistant variety. Using gene engineering technology to improve the quality of variety directionally is efficient approach.Chitinase could decompose fungal cellwall, thereby restrain the growth and multiplication of fungi. Antifungal peptide is a new type of antifungal substance, which have wide antifungal spectrum and high antifungal activities.In this study, binary gene expression vector contain CHI gene from balsampear(Momordica charantia) and AFP gene from motherwort Herb (Leonurus heterophyllus) were transformed to maize via Agrobacterium tumefaciens and micro particle bombardment. The results are as the follows:1. The effect of different size of immature embryo on the induct rate and quality of calli was studied .The explant size between 1.0-2.0mm can inducted higher quality calli than other size,calli induction ratio reached 90%.MET 2mg·L~- promote rooting, and can easily survive.2. Optimization of the influencing factors of maize transformed by Agrobacterium tumefaciens. The effect of beforehand cultivation time, tinging time and different acetosyringone (AS) concentration on transformation efficiency were studied. The results demonstrated a appropriate condition, which contain a beforehand cultivation time of 6-9 d, tinging time of 10min and 100uM AS in co-culture, transformed efficiency reach 24.25% under this parameters. In the micro bombardment transformation method, bombarding distance of 9cm produce a higher transformed efficiency than that of 6 cm, transformation efficiency was about 35% under these parameters.3. Checkup of transgenic plantsGus assay: herbicide-resistant calli and leaves transformed plants appeared blue color, which demonstrate target genes had been transmited into genome and expressed PCR analysis: Genome DNA was distilled from transformed plants and control for PCR amplification. Target fragment about 401bp were amplicated from transformed plants, but none from control, which showed that target genes had integrated into plant genome.Herbicide-resistant experiment: Leaves of transgenic plants and control were plug into medium with 200mg·L~- PPT, Leaves of control became yellow 5 d later, but Leaves of transgenic plants keep green still.4. Primary analysis on the desease-resistant of transgenic plantsTransferring the Pathogens of Exserohilum turcicum onto the leaves of transgenic maize plants in vitro.3 days later, Lesions appeared on non-transgenic plants, however the leaves of transgenic plants keep healthy still.Both the single CHI and binary genes CHI-AFP showed the desease-resistance.