Studies On Ca Signal Transduction And Protein Expression Of Longan (Dimocarpus Longan Lour.) Under Acid Rain Stress

One age seedlings of longan were selected in this study. The responses of the content of CaM, the activities of H⁺-ATPase and Ca²⁺-ATPase to acid rain stress, and the specific protein expression were studied. The exogenous Ca²⁺ regulation and the mechanism of receiving and transferring the stress signals were probed. The main results were as follows:The procedures of quantitative determination of calmodulin (CaM) by ELISA were optimized first. The optimal coating concentration of calmodulin (CaM) was 5μg/ml, the optimal dilution factor for primary antibody was 1:3000 and the optimal dilution factor for sample was 40.The content change of calmodulin (CaM) was influenced by stress intensity and duration. The content of calmodulin (CaM) increased and decreased repeatedly during stress, but the variation amplitude decreased gradually while stress duration prolonged. The activities of H⁺-ATPase and Ca²⁺-ATPase were also influenced by stress intensity and duration. The activities of Ca²⁺-ATPase and H⁺-ATPase were very sensitive to acid rain, and which were inhibited under high stress intensity. The reason why the activities of Ca²⁺-ATPase and H⁺-ATPase decreased was that the high H⁺ concentration destroyed Ca-CaM signal system. The results lead to the stress resistance of plant decreased gradually.At the present of the 10mmol/L Ca²⁺, the content of calmodulin (CaM), the activities of Ca²⁺-ATPase and H⁺-ATPase were higher than which without Ca²⁺. The EGTA reduced the content of calmodulin (CaM), and inhibited the activities of Ca²⁺-ATPase and H⁺-ATPase. The 2mmol/L CPZ made the same effect as EGTA. The results showed that the Ca-CaM signal system played a role of transferring the acid rain stress signals, the activity of Ca²⁺-ATPase and H⁺-ATPase were activated and regulated by CaM, and the exogenous Ca²⁺ can enhance the signal transferring.The soft Quantity one and PDQuest were used for imaging and analyzing 1-D and 2-D electrophoresis gel images in this research. The softs can make the clear background and keep data integrity. The molecular weight (Mr) of protein bands was calculated by Quantity, and the soft also calculated the similar rate of each lane in SDS-PAGE images. The PDQuest calculated the molecular weight (Mr) and isoelectric point (pI) of each protein component in 2-DE images, and the amount of protein components were also done. The specific protein components were found by PDQuest. SDS-PAGE technique was applied to compare and analyze the specific protein expression of longan leaves during simulated acid rain stress. The results showed that there was a protein band specifically expressed under pH2.5 acid rain stress for 15 days, whose molecular weight is about 23KD. The 17KD protein specifically expressed after stress for 5 days. Compared with pH2.5, the pH3.5 acid rain made a less influence to protein expression, but the differential expression was raised along with the enhancement of acid rain stress.The 2-DE technique was applied to compare and analyze the changes in protein component during stress. The major components of proteins were generally similar at different pH value acid rain, but some changes were also observed. There were 6 new specific proteins were expressed. The protein C₄(20.2KD pI6.0) expressed in all pH values acid rain during stress period. The protein F(24KD pI6.35) expressed under pH2.5 acid rain stress for 15 days, and then expressed in all pH stress for 20 days. The expression of proteins including B₃(22.3KD pI6.35), B₄(23.5KD pI6.5), C₅(21.8KD pI5.6), C₆(20.2KD pI5.6) are related to the acid rain pH value and stress intensity. The protein B₄ only expressed in groups including exogenous Ca²⁺, showing that the exogenous Ca²⁺ involved in the expression of B₄.The research found that there were 4 proteins expression increased under the treatment of EGTA and CPZ. These proteins might be relative to the Ca-CaM system of Iongan in response to acid rain stress.