Upland Transformation Of PPA Gene By Agrobacterium

Due to the shortage of water resource,rice production is restricted.So upland rice as a less water and drought resistant variety has good development and application prospects.As pests is an important factor to decrease the yield of rice,the use of transgenosis to cultivate the upland rice with insect resistance,has economic benefits and ecological benefits.PPA gene has insecticidal physiological activity,whose application has a bright prospect in insect-resistant transgenic crop breeding.This research chose “Handao 2” as the experimental material,with Bar gene as a biomarker for screening,and then transformed the insect-resistant gene of PPA by Agrobacterium-mediated transformation.Finally,this study optimized the conversion system,and produced the transgenic upland rice plants,who has the herbicide resistance and the activity of insect-resistant.To get T₁ generation plants which have resistance of herbicide,0.5 mg/mL of the glufosinate was spray in field.In the laboratory,T₂ generation transgenic lines were homozygous screening,lines with herbicide resistance were identified by PCR to find homozygous lines.In this study,Handao2 with PPA gene positive strain was identified by Southern hybridization.There are many factors that effect Southern hybridization,such as the digestion system,DNA concentration of the sample,concentration of the probe,hybridization conditions.The results showed that,adding 15?g of DNA,90 U endonuclease to the 60 ?L digestion system and digesting 15-16 h is the most suitable.Adding 150 ng of probe to 5 mL hybridization solution in the hybridization system is most suitable.Southern blot analysis showed that,PPA gene integrated into the genome of Handao2 in the form of multiple copies.