Two-dimensional Electrophoresis Analysis Of Proteins Related To The Resistance Of Jujube Wicthes' Broom Disease

Chinese jujube(Ziziphusjujuba Mill.)is a kind of important and specific fruit tree in China.Jujube witches'-broom disease(JWB),caused by phytoplasma,is one of the most destructive diseases in Chinese jujube..Through 10 years' study,an excellent new cultivar with very high resistance to JWB,named "Xingguang",was bred up and released in 2005. It was used to screen the proteins related to the resistance of JWB by two-dimensional gel electrophoresis(2-DE)and mass spectrum analysis technology.The main results were as follows:1 An optimized two-dimensional gel electrophoresis method of Chinese jujube was established.The TCA-acetone method,ammounium acetate methanol method,and improved Tris-HC1 method were compared in this study.The result showed that ammounium acetate methanol method which could throw off the rich polysaccharides and pigment of jujube was propitious to total protein isolation of jujube phloem.The feasible capacity of protein was 380μl(about 200 ug),the best balance time was about 30min～40min,and the proper SDS-PAGE voltage was about 200V. Silver-stained method was confirmed to be the better dyeing method for jujube phloem protein compared with coomassie R-250 dyeing method.The pI of jujube phloem protein was ascertained mostly between 4～7.Using the established protein isolation and two-dimensional gel electrophoresis system,total protein from branch phloem was separated by pH4～7 IPG and silver-stained method,the 2-DE gel was analyzed by PDQuest.The results indicated higher repetition and differentiation. 500～700 protein spots were detected.2 The resistance-related proteins of Jujube wicthes' broom disease in Ziziphus jujuba Mill.were analyzed.Through SDS-PAGE analysis,there were no obvious difference among 'Xingguang' scions 70d and 90d after graining on dieased and healthy trees,but there were three protein bands' expression capacity(MW 31.2 kDa,30.4 kDa,29.0 kDa)increased remarkably in the scions 110d after grafting in the dieased trees. Through 2-DE analysis,an expression capacity area(MW 27～32KD,pI4.2～5.0)at acid end increased gradually in 'Xingguang' scions 90d and 110d after grafting on diseased tree.This result is consistent with the SDS-PAGE analysis.The range of resistance-related protein of JWB was MW 27～32KD,pI4.2～5.0.3 Three better homology peptide sequence were screened out.About 10 specific protein spots were detected,and only 3 of them showed some similarity with known proteins in the database.They were soluble NSF attachment protein,glyceraldehyde-3-phosphate and clathrin adaptor complexes medium subunit family protein.The other 7 proteins were unknown proteins.The function of identified proteins related to the resistant mechanism of JWB was analyzed and discussed.