Expression Characteristics Of Differentially Expressed Proteins In Response To Pb And Zn Combined Stress In Carex Putuoshan Root

A new lead hyperaccumulator plant named Carex putuoshan was founded in Mount Putuo lead-zinc mine area at the county of Hanyuan in Ya’an city. As a special Carex, Carex putuoshan has strong reproduction ability, high biomass, barren-tolerance and other characteristics in the lead and zinc compound polluted soil. In order to further explore the plant’s lead hyperaccumulation mechanism, and to look for the lead hyperaccumulation response protein components, we investigated the differentially expressed proteins of Carex putuoshan under Pb and Zn combined stress via two-dimensional gel electrophoresis, mass spectrometry and bioinformatics technology according to the principle of comparative proteomics. The results showed as follows:(1) We established a new two-dimensional gel electrophoresis system that suitable for Carex putuoshan roots’ protein extracting, and improved the traditional Tris-HCL/TCA-Acetone precipitation. At the same time, we found a better method to extract the protein of Carex putuoshan roots and optimize the conditions of isoelectric focusing. Subsequently, we acquired a rich and clear two-dimensional gel electrophoresis picture of Carex putuoshan roots’ total proteins which include 376 protein spots.(2) We used mass spectrometry to identify the 10 most obvious proteins of Carex putuoshan roots’ under the compound stress of lead and zinc. After applying Pb and Zn combined stress, the expression of 9 protein spots (including 7 different proteins,2 identical proteins,1 unknown protein) in Carex putuoshan root was found to be significantly up-regulated. They are involved in glyceraldehyde-3-phosphate dehydrogenase (GAPDH), malate dehydrogenase (MDH), fructose-1,6-bisphosphate aldolase (FBA), enolase,6-phosphogluconate dehydrogenase (G6PDH), isoflavone reductase (IFR), and phytochelatin synthase (PCS).(3) By subcellular location and bioinformatics analysis of protein, we found that the proteins mainly located in the cytoplasm. The bioinformatics analysis of protein showed that each protein has a multiple function, They mainly participate in redox process and carbohydrate metabolism, many enzymes have the redox enzyme activity.Our research indicated that the process of lead hyperaccumulation was a large enzyme activity system, mainly involved in Carbohydrate and energy metabolism, the most active enzyme was hexose lyase and pentose phosphate enzyme system. GAPDH and G6PDH were especially outstanding, therefore, they can be the key response element protein of Carex putuoshan roots under the compound stress of lead and zinc. The changes in GAPDH and G6PDH expression can be used as an important index for the lead hyperaccumulation of Carex putuoshan, and their protein molecular weight were 14.7 KDa and 35.9 KDa.