The Proteomic Analysis Of Plasma Membrane Of Rubber Tree (Hevea Brasiliensis Muell.Arg) Bark Under Ethylene Stress

Natural rubber is mainly produced by rubber tree's bark,ethylene stress would promote the process of production and release of rubber latex.There are a variety of hypothesis and mechanisms that ethylene can help increasing production of rubber latex,but mechanism from plasma membrane proteomic level is rarely reported.In this study,the aqueous two-phase partition system was employed to obtain plasma membrane from 2 years budding clones 'SCATC 7-33-97' rubber tree bark.By the means of mark enzyme activity detection,electron microscope detection and protein concentration detection,the effect of different two-phase system for plasma membrane was evaluated,and optimum plasma membrane extraction method of the rubber tree bark was established.After being treated by Brij58 turnover eluent,plasma membrane proteins were lysised by SDS lysis buffer,Urea lysis buffer and Methanol lysis buffer,then liquid chromatography mass spectrometry?LC-MS?was used to identify rubber tree bark plasma membrane proteins,and by comparing the number of lysised plasma membrane proteins,the most suitable method for rubber tree cortex membrane protein lysis buffer was chosen.The extracted membrane proteins were then analyzed by label-free quantitative technique to quantitative different expressional proteins under ethylene stress,and studied metabolism and signaling pathways by bioinformatics methods,which on the level of plasma membrane proteome revealed the mechanism of promotion of rubber production under ethylene stress.The expression of H⁺-ATPase was confirmed by QRT-PCR,which was consistent with the protein expression level.The results are as follows:1.Optimization of extracting method for the bark plasma membrane of rubber tree,6.4%?W/W?dextran and polyethylene glycol concentration,and 5 mM KCl concentration was the best two-phase system for the separation and purification of the rubber tree bark's plasma membrane.The crude microsome was purified three times by the two-phase system given the purity of 78.05%,at the same time the content of mitochondrial membrane and chloroplast membrane was reduced to 7.18%and 1.08%,respectively.The contamination of the vacuolar membrane was reduced to 1.08%.After purification the plasma membrane composition was single,the vesicles were dispersed uniformly with consistent size.Brij58 turnover eluent can remove some non-specific binding proteins,weak-interaction proteins and intracellular soluble proteins,the proportion of plasma membrane proteins is increased.2.Screening of the lysis buffer to clevage of plasma membrane proteins.SDS lysis buffer was suitable for the dissolution of rubber bark plasma membrane proteins.After being treated by SDS lysis buffer,2685 proteins was totally identified,in which the number of membrane proteins and plasma membrane proteins was 1726 and 329,respectively.3.Protein expression level analysis of plasma membrane proteins after ethylene stress.24 h or 48 h after being treated with ethylene,the proteins with Fold-change>2 was selected,in which the number of up-regulated proteins was 76 and 106,48 and 63 proteins was down-regulated.Those proteins were further analyzed by the means of bioinformatics to study the mechanism of ethylene stress involved in the material transportation,sugar metabolism,water transportation,nitrogen metabolism,intracellular pH adjustment,cell wall synthesis and structure change,signal transduction,etc.4.Confirmation of the consistent of expression between gene and protein levels of H⁺-ATPase.After ethylene stimulated,H⁺-ATPase with differential expression was selected for further QRT-PCR analysis to comfirm gene expression level.The results showed that H⁺-ATPase?scaffold0127₃17412,scaffold1940₃5256?was significantly up-regulated,while some H⁺-ATPases?scaffold0012₅88005,scaffold0031₁905157,scaffold0206₁539650,scaffold0052₈19927,scaffold0073-380500?were slightly or not up-regulated,confirmed the expression level of H⁺-ATPase.