2-DE PROTOCOL OPTIMIZATION AND EVALUATION FOR PROTEOME ANALYSIS OF GENUS CLEMATIS TAXA (RANUNCULACEAE)

摘要

An approach was conducted to optimize two-dimensional gel electrophoresis (2-DE) method for leaf proteome analysis ofgenus Clematis species, as a molecular approach to explore its taxonomy and differentially expressed genome patterns. Duringestablishment and optimization of protocol we extracted proteins by three extraction protocols, viz., phenol-SDS (PS) method,TCA/acetone (TA) method and lysis buffer (LB) method, and PS was the best one with 2.35±0.05 μg protein yield. For proteinsolubilization two lysis buffers (LB-1 & LB-2) were prepared, used and comparatively LB1 depicted better resolution. Proteinswere by quantified by the Bio-Rad protein assay (Hercules, CA, USA) with bovine serum albumin as standard and purified by2-D clean-up Kit (Amersham Biosciences). 2-DE analysis was conducted on pH 3~10, non-linear gradient strips (24cm) as firststep, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 13% polyacrylamide gels as the secondphase. For spot visualization gels were stained for with silver stain. The gels were scanned using Powerlook 2100XL scannerand gel images were analyzed by ImageMaster 2-D Platinum. Validation of experiment was performed by measuring analyticalvariance (AV) and biological variance (BV) for replicate spots. AV was calculated for 60 protein spots present in three replicate2-DE gels of the same protein extract and BV was determined for the same protein spots from independent tissue extractscorresponding to leaves from different plants, or the same tree at different orientations or sampling times during a day. Valuesof 26% for the analytical variance and 58.6% for the biological variance among independent sampled species were obtained.This provided a threshold values for the evaluation of protein expression changes in comparative proteomic investigations withthis species. Some spots were selected and subjected to liquid chromatography mass spectrometry (LC-MS) for identificationpurpose. Due to absence of Clematis DNA or protein sequences databases, FASTA and BLAST similarity searches wereperformed against other plant species databases were used for protein identification. The significance of 2-DE proteomeanalysis in predicting evolutionary trend of Clematis (liana) species and its potential significance in taxonomic identification forTraditional Chinese Medicine (TCM) pharmacopeia is described.