Differential Proteins Expression Induced By Aluminum Treatment In Leaves Of Different Eucalyptus Clones

Eucalyptu is an important tree species in South China, especially in Guangxi Province. In recent years, the tendency of acidity and increasing Al3+in soil with rich aluminum has been more and more serious in South China, which would be one of the potential harmful factors to affect the growth of Eucalyptus.The research on aluminum resistance mechanism in fast-growing Eucalyptus is greatly significant to improve the productivity of Eucalyptus plantation and Eucalyptus plantation forest sustainable management.The E. grandis×E. urophylla clone GLgu9and GLgul2, E. urophylla clone GLu4and E. wetarensis×E. camaldulensis clone GLwc3were treated by in Al3+stress sourced from AICl3·6H2O in acidic environment in this study. Based on two-dimensional electrophoresis technology optimization of Eucalyptu leaves, the differential protein expression of4Eucalyptus clones leaves were analyzed.The main research results were summarized as follows.(1) The two-dimensional electrophoresis technics was optimized by different methods of protein extraction. The results showed that phenol extraction was suitable for Eucalyptus leaf protein extraction. Compared to the direct extraction method and improved acetone precipitation, the method of phenol extraction was more effective to extract protein from leaves, which generated more protein spots with good indexes of distribution—balance, round, less tail, less impurities and more clear on2-D map for identification of proteins by the mass spectrometry.(2) The better lysis buffer contained7M urea,2M thiourea and4%Chaps. Adding80mmol DTT10μl enhanced dissolution effect; When600μμ1ysis buffer with50mg protein powder, protein concentration was up to3.97μg/μl.(3) There were round and clear protein spots and Hess stipes on2-D map while protein sample amounted to1200μg on the24cmIPG strip with pH4-7. The results met the demand of two-dimensional electrophoresis analysis. When protein sample amounted to600μg, protein spots were few.When the sample amounted to900ng, there were about250protein spots on the2-D map. When the sample quantity increased to1200μg, protein spots reached around850that were suitable for software analysis and mass spectrum identification.(4) The two-dimensional electrophoresis technology was used to identify the differential proteins in the leaves of GLgu9, GLgu12, GLu4and GLwc3under aluminum treatments.The sample gel of GLgu9obatained14different spots,4spots were up-regulated and10spots were down-regulated;The sample gel of GLgul2obatained38different spots,22spots were up-regulated and16spots were down-regulated;The sample gel of GLu4obatained33different spots,16spots were up-regulated,6spots were down-regulated,1spots disapperead and10novel spots appeared;The sample gel of GLwc3obatained13different spots,2spots were up-regulated,2spots were down-regulated,6spots disapperead and3novel spots appeared.(5) Different protein were deteced by MALDI-TOF-TOF tandem mass spectrometry and database retrieval.The protein was mainly divided into the following types:(a) Enzymes and relevant subunits related to photosynthesis and carbon reaction: the chlorophyll a/b binding protein, ribulose1,5-bishosphate carboxylase/oxygenase, ribulose1,5-bishosphate carboxylase/oxygenase large subunit;(b) Enzymes related to sugar metabolism:glyceraldehyde3-phosphoric acid dehydrogenase;(c) Enzymes of oxidation resistance function and signal transduction:2-cysteine peroxidase, oxygen increase protein;(d) Enzymes related to protein synthesis and metabolism: plastid glutamine synthetase.The research of Eucalyptus protein expression under aluminum treatments will provide theoretical basis to study on the molecular response mechanism in Eucalyptus to aluminum. And it will be helpful to choose Eucalyptus clones with resistance to aluminum as a technical reference, furthermore, to make genetic improvement for getting higher productivity of Eucalyptus stands.