Optimization Of Direct Transformation Of Minimal Linear Gene Cassette And Its Transient Expression

Direct transformation of minimal linear gene cassette has been widely used in plant gene transformation,with the advantages of easily operation,high transformation efficiency and wide range host transformants.Exogenous gene could be integrated into the host DNA, expressed and stably inherited to progenies.Direct transformation of linear gene cassette has ever been used in stable transformation,however,it remains unconvinced because of its insufficiency and unrepeatability.Using genes smGFP and GUS as two linear gene cassettes, the present work aims at evaluating different physicochemical co-factors,including surfactants,osmotic and calcium ion,and imaging the delivery and localization of the linear gene cassettes through transient expression in vivo.It was expected to establish a direct transformation system for transient expression with minimal linear gene cassette,and may also contribute to the potent application in stable gene transformation.Binary expression vector of smGFP and GUS were constructed to transform onion epidermal cells mediated via Agrobacterium-mediated transient transformation system.The transformation efficiency of gradient concentration of transformation co-factors,including CaCl₂,6-BA,2,4-D,Silwet L-77,Dow CorningQ2-5211,as well as osmotic treatement were evaluated.The results showed that each physicochemical factor had active effects on Agrobacterium-mediated transformation and transient expression,and the optimum concentration of was 0.5mol/L of CaCl₂,0.2mg/L of 6-BA,0.2 mg/L of 2,4-D,0.1‰Silwet-77, and 0.1‰Dow Coming Q2-5211,respectively.Compared with the expression intensity of Green Fluorescent Protein and quantity of punctate blue stain,Agrobacterium infiltration was the optimal treatment in improving transformation efficiency.The transient expression of liner smGFP/GUS was evaluated through observation of fluorescent/stain intensity at 30min.2h.6h,12h,36h,48h post transformation.Compared with the expression intensity of GFP and quantity of punctate blue stains,Agrobacterium infiltration obtained the higher transformation efficiency compared with the co-factor treatements.Meanwhile,the delivery and orientation of linear transformation cassettes were traced through fluorescent indicators of FITC or SYBR Green I.The entry rate of the linear transformation cassette into nuclei was quantitatively measured by flow cytometry.The results showed that the efficiency of transient expression in lower onion epidermal was dramatically increased after 15-20min hypertonic treatment followed by 100ng/mL linear DNA co-culturvatied in MS liquid medium for 1.5-2h,suggesting its feasibility in direct transformation of linear gene cassette.The linear smGFP gene cassette was then transformed into tobacco leaf callus and To trangenic seedlings were further identified by the PCR,RT-PCR and dot blotting.High regeneration rate as well as higher transformation efficiency was achieved.Our study first reports the direct hypertonic transformation of liner gene cassette in transient and stable expression.