Isolation Of Mesophyll Protoplast From Arabidopsis Thaliana And The Setup Of Transient Expression Assay System

Arabidopsis has great advantages and plays an important role in bioresearch as a modal plant.This study chose Columbia wild type Arabidopsis as material to isolate mesophyll protoplast with enzymolysis method and deliver exogenous gene into protoplast to express transiently using PEG-mediated method.We mainly investigated the effect factors on isolation of Arabidopsis mesophyll protoplast and transformation and abtained the optimum conditions for protoplast isolation.Furthermore,we set up a transient expression system based on Arabidopsis mesophyll protoplast.The main results are as follows:1.The optimum combination and concentration of enzyme solution for protoplast isolation.We isolated Arabidopsis mesophyll protoplast with different combination of enzymes:cellulase R-10,hemicellulase,pectinase,macerozyme R-10 and drislease.The best yield reached 9.55×10~6 cells/gFW with few cells broken down while incubating young leaves in the CPW solution containing 1.5%cellulase R-10 and 0.5% macerozyme R- 10.2.The best conditions for digestion.We analysed the factors of digestion,including incubation time and the PH of enzyme solution.The best yield of protoplast (11.42×10~6cells/gFW) and the highest viability(90.2%) were abtained at PH 5.8,28℃in dark,incubating for 4 hours.3.The stability maintenance and viability analysis of protoplast.We used mannitol as osmotic regulator to keep protoplasts stable and analysed the cell viability with FDA-staining.The result showed the mesophyll protoplast can keep stable with spherical shape in enzyme solution which containing 0.4M mannitol,the cell disruption was minimized and cell viability was highest(90.1%) under such condition.4.Set up of transient expression system.We studied the transient expression of SIP (small and basic intrinsic protein) and ARP6(actin related protein 6).Genomic sequences for SIP and ARP6 were cloned from Arabidopsis RNA by RT-PCR.The resulting fragments(SIP 720bp;ARP6 1200bp) were ligated into the SpeI-XhoI and BamHI sites of the vector pA7-YFP respectively,then transiently expressed in Arabidopsis mesophyll protoplast by PEG-mediated method.We investigated the effects of PEG concentration, plasmid DNA content,protoplast numbers on transformation.The results showed that efficiency of transformation was raised with the increased PEG concentration and DNA content.The highest efficiency reached 69.37%when the incubating mixture contained 20%PEG,20μg plasmid DNA and 2.5×10~4 protoplast cells.