Study On Arabidopsis Protoplast Isolation And Phytase Gene OsMINPP Transient Expression

It is important for research the model plant Arabidopsis protoplast isolation and transient gene expression in biology study. It is analyzed the leaf and root derived protoplast isolation method use wild type Arabidopsis co10 as material, optimized transient expression system using mesophyll protoplast and root protoplast, analyzed the relationship between phytohormones and OsMINPP expression, and discussed OsMINPP cellular localization preliminarily by using the system. Main results are as follows:1.Peeling away lower epidermis of Arabidopsis leaf by paper tape reduced protoplast broken rate. By using different leaf treatment method, it was found to reduce protoplast broken rate to 1.8% and reduced by 92.1% by paper tape method compared with regular cutting strip method.2.CaCl2 treatment improved Arabidopsis mesophyll protoplast yield.By supplying WI,W5 and MMG with 0-50 mmol/L CaCl2, it was found to improve the highest protoplast yield when lOmmol/L CaCl2 was added in WI.3.IAA and GA3 treatment promoted protoplast yield. By treated phytohormones with spraying plant and injecting enzyme solution,protoplast isolation process enhanced by IAA and GA3, and yield improved by 46.9% and 48.3% respectively compared with contrast.4.Obtained the best protoplasts isolation condition for root. By optimized factors influencing protoplasts isolation, a best yield was reached (9.23×106 number/gFW) under the condition that 1.5% cellulase R10 combined with 0.4% macerozyme RIO hydrolyzed cell wall,0.4 mol/L mannitol maintain osmotic pressure and centrifuge at 1600 round per minute (200 g) for purification.5.Obtained a transient expression system using Arabidopsis root protoplast. By optimized factors influencing transient expression, a high expression efficiency was achieved (62.24%) after 30% PEG,25μg plasmid and 7.5×104protoplasts incubated for 5 min.6.GA3 improved OsMINPP promoter activity. After adding 10-6 mol/L IAA,BL,ABA and GA3 into the transient expression system incubation for 12h, the GUS activity was detected. The results showed that GUS activity of GA3 group was 2.14 times of contrast group and thus improved the expression of OsMINPP promoter.