Establishment Of The Techniques For Protoplast Isolation And Protein Subcellular Localization Of Ammopiptanthus Mongolicus

protoplast is one of the receptors for gene transient expression like protein subcellular localization. Therefore the establishment of the techniques for the isolation and purification of plant protoplasts is an important prerequisite for the related researches. Ammopiptanthus mongolicus is an important plant species with strong resistance to adverse environments. In the present study, we firstly established the techniques for the protoplast isolation and gene transient expression for this plant. Then, we performed the subcellular localization analyses of two stress-related genes, namely AmDREB1F and AmDREB2 cloned from A. mongolicus with the established techniques, by using the protoplast transient expression system of the model plant Arabidopsis as reference. The main results are as follows:(1) Using the young leaves from A. mongolicus aseptic seedling as material and the one-step enzymolysis method, a preliminary technique for the protoplast isolation and purification were established:the enzyme solution was composed of 3% cellulose,0.5% macerozyme,0.3% hemicellulose,9% mannitol and 0.15% CaCl2, pH 5.8; the enzymatic hydrolysis was conducted under 25℃, dark, and 40 rpm for 14 hours. The protoplast purification was performed using the precipitation method in W5 solution at 700 rpm for 5 min.(2) The coding region cDNA of AmDREB1F, AmDREB2 or AmCAT gene was successfully inserted into the plant transient expression vector pBI-GFP, and three fusion protein expression vectors, namely pBI-AmDREB1F-GFP, pBI-AmDREB2-GFP and pBI-AmCAT-GFP, were obtained.(3) Using the PEG-mediated transformation method, the constructed expression vectors were introduced into the protoplasts from both Arabidopsis and A. mongolicus seedlings, respectively. As a result, the AmDREB1F and AmDREB2 proteins were localized in the nucleus, while the AmCAT protein may exist in peroxisomes.