Gene Transformation Of Sugarcane With RNAi To Sugarcane Mosaic Virus And Yellow Leaf Virus Via Biolistics And Molecular Identification Of Transgenic Plants

Sugarcane is an important sugar-yielding crops and energy crops,sugarcane main diseases such as sugarcane mosaic disease and sugarcane yellow leaf disease restrict the development of sugarcane industry,at the same time,the complexity of sugarcane genetic background,the long hybrid process cycle by cross breeding and the detoxification of the tissue culture has such problems as the big workload and be virus reinfection.These restrict to gain disease resistance by cross breeding and gain virus-free seedling by tissue culture.So the application of genetic engineering technology become an effective way to improve sugarcane properties of resisting to diseases and pests.This research use expression vector of bivalent interference pART27-MC1Y,pART27-MC2Y with sugarcane mosaic virus HC-Pro gene and sugarcane yellow leaf virus CP gene,and pART27-MPY with sugarcane mosaic virus CP gene and sugarcane yellow leaf virus CP gene which has been built and saved in the laboratory.On the base of verifying the expression vector integrality and establishing the optimal screening concentration of G418 on sugarcane variety FN15 in callus differentiation period and differentiated seedlings growth period,then using biolistics transforms three expression vectors to sugarcane variety FN15 respectively and detecting the resistible sugarcane variety FN15 regenerated plants with genetic transformation by genetically modified molecular identification,and obtaining the transgenic plants.The main research achievements of this paper:1.Verifying pART27-MC1Y,pART27-MC2Y and pART27-MPY expression vectors integrallty by PCR and restriction endonuclease digestion,then transform sugarcane variety FN15 callus by biolistics.2.Establishing resistance screening concentration of G418 is 35mg/L in the sugarcane callus differentiation period and 40mg/L in the sugarcane differentiated seedlings growth period by the gradient filter of G418 concentration.3.Gaining twelve transgenic sugarcane plants by PCR detection,confirming the copy number in five transgenic sugarcane plants with RNAi bivalent interferential structure of sugarcane mosaic virus CP gene and sugarcane yellow leaf virus CP gene by real-time fluorescence quantitative PCR,and making use of semi-quantitative and quantitative PCR to detect the expression of the transgenic sugarcane plants.The result show that the target gene had been expressed in the transgenic sugarcane plants.And utilizing the method of quantitative PCR to analy the relation between transgenic copy number and expression quantity,the result show that the copy number is between 3.15 to 5.93 in the transgenic sugarcane plants without the obvious corresponding relation to relative expression.