Study On Differentially Expressed Proteins Of Chloroplast From Transgenic Tomato Mutant With HBsAg Gene

In a previous study about the transgenic tomato mutant with HBsAg gene (N244), we found that N244showed obvious physiological alterations compared with nontransgenic tomato (CK). In particular, the leaves of transgenic plant were fleshy and dark. The further study showed that the chlorophyll content, chloroplast number, ultrastructure of the chloroplast of N244had significant difference compared with CK. To explore the mechanism of mutation, first, comparison of photosynthetic characteristics between N244and CK was studied, and then their chloroplast proteins were separated by two-dimensional electrophoresis. The function of differentially expressed proteins was analysed combined with the photosynthetic result. The major results were as follows:1. The light compensation point, light saturation point, net photosynthetic rate, apparent quantum efficiency and dark respiration rate of N244were respectively11.18μmolm-2s-1.1690.64μmolm-2s-1,19.23μmolCO2m-2s-1,0.0528and1.1371μmol CO2m-2s-1; The corresponding data of CK were respectively14.70μmolm-2s-1,657.20umolm-2s-1,10.14μmolCO2m-2s-1,0.0725and1.1324μmolCO2m-2s-1. The light compensation point and net photosynthetic rate of N244was significantly greater than CK.2. The chloroplast proteins of the mutant N244were analyzed by two-dimensional difference gel electrophoresis. The intact chloroplasts were isolated from tomato leaves by differential centrifugation combined with sucrose density gradient centrifugation. The TCA-acetone precipitation was applied to extract chloroplast protein.200μg protein was loaded onto each IPG strip (18cm, pH4-7), and the second dimension was separated by12.5%SDS-PAGE electrophoresis followed by silver staining. At last, electrophoresis pattern with a good repeatability was obtained.3. The protein pattern was analyzed the software Image Master2D Platinum6.0, nearly180protein spots were detected. Some differentially expressed protein spots were identified by MALDI-TOF-MS analysis and NCBInr searching,which are ATP synthase F1subunit1、 chlorophyll A/B binding protein、Polyphenol oxidase F.ATP synthase beta subunit.ATP synthase CF1beta chain. catalase isozyme1and some hypothetical proteins.The functions of the differentially expressed proteins were analyzed.