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| 1 | Cloning and expression of AtPLC6, a gene encoding a phosphatidylinositol-specific phospholipase C in Arabidopsis thaliana显示文摘A full-length cDNA clone corresponding to a putative phosphatidylinositol-specific phospholipase C (PI- PLC) was isolated from Arabidopsis thaliana by screening a cDNA library and using RT-PCR strategy. The cDNA, des-ignated AtPLC6, encodes a putative polypeptide of 578 amino acid residues with a calculated molecular mass of 66251.84 D and a pI of 7.24. The sequence analysis indicates that the polypeptide contains X, Y, EF-hand and C2 domains. The overall structure of putative AtPLC6 protein, like other plant PI-PLCs, is most similar to that of mammalian PLCd. The recombinant AtPLC6 protein expressed in E. coli was able to hydrolyze phosphatidylinositol 4,5-biophosphate (PIP2) to generate inositol 1,4,5-trisphate (IP3) and 1,2-diacylglycerol (DAG). The protein hydrolyzes PIP2 in a Ca2+-dependent manner and the optimum concentration of Ca2+ is 10 mmol/L. These results suggested that AtPLC6 gene encodes a genuine PI-PLC. Northern blot analysis showed that the AtPLC6 gene is expressed at low level in all examined tissues, such as roots, stems, leaves, flowers, siliques and seedlings under normal growth conditions. The gene is strongly induced under low temperature and weakly induced under various stresses, such as ABA, high-salt stress and heat. These results sug-gested that AtPLC6 might be involved in the signal-trans- duction pathways of cold responses of the plants. | XUXiaojing CAOZhixiang LIUGuoqin MadanK.Bhattacharrya~ RENDongtao | 2004 | Chinese Science Bulletin2004，49，6： | 5 |
| 2 | Prokaryotic expression and characterization of a pea actin isoform (PEAc1) fused to GFP显示文摘Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells,it is difficult to purifyeach actin isoform in sufficient quantities for analysing itsphysicochemical properties. In the present study, apea(pisum Sativum L.)actin isoform (PEAc1)fused to His-tag at its amino terminus and GFP(green fluorescent protein)atits Carboxyl terminus were expressed in E. coli in inclusionbodies. The fusion protein (PEAc1-GFP)was highly purifiedwith the yield of above 2 mg/L culture by dissolving inclu-sions in 8 mol/L urea,renaturing by dialysis in a gradient of urea,and affinity binding to Ni-resin. The purified mono-meric PEAc1-GFP could efficiently bind on DNase I andinhibit the latter抯 enzyme activity. PEAc1-GFP could po-lymerise into green fluorescent filamentous structures(F-PEAc1-GFP),which could be labelled byTRITC-phalloidin,a specific agent for observing microfila-ments. The PEAc1-GFP polymerlzation curve was identicalwith that of chicken skeletal muscle actin. The critical con-centration for PEAc1-Gfp to polymerise into filaments is 0.24 μmol/L.The F-PEAc1-GFP could stimulate myosinMg-ATPase activity in a protein concentration dependantmanner (about 4 folds at 1 mg/mL F-PEAc1-GFP). The re-sults above show that the PEAc1 fused to GFP retained theassembly characteristic of actin, indicating that gene fusion,prokaryotic expression, denaturation and renaturation,andaffinity chromatography is a useful strategy for obtainingplant actin isoform proteins in a large amount. | ZHANGShaobin RENDongtao XUXiaojing LIUGuoqin | 2004 | Chinese Science Bulletin2004，49，9： | 2 |
| 3 | Measurements of leucocyte membrane elasticity based on the optical tweezers显示文摘A 1-mm in diameter polystyrene bead coated with lectin is trapped by optical tweezers which is formed by a focused laser beam (740 nm). A leucocyte adhered to the bottom of sample cell is chosen to close to the trapped bead. When their stable combination is confirmed, the leucocyte is displaced by moving the sample stage. A tether is then formed between the trapped bead and the membrane. The force acting on the tether is measured by a detector equipped on the optical tweezers system. The deformation of the membrane is diverse in different contact conditions. When the contact area is small, the tether is very thin and the force on it is 7.4 pN (10-12 N), while the tether is much thicker un-der a larger contact area and its corresponding force is about 14 pN. It is presumed that the latter is related to the defor-mation of cytoskeleton. | GUOHonglian CAOQinhong RENDongtao LIUGuoqing DUANJianfa LIZhaolin ZHANGDaozhong HANXuehai | 2003 | Chinese Science Bulletin2003，48，5： | 1 |
| 4 | Constitutive activation of AtMEK5,a MAPK kinase,induces salicylic acid- independent cell death in Arabidopsis thaliana显示文摘AtMEK5 DD is an active mutant of AtMEK5, a MAP kinase kinase in Arabidopsis. Induction of AtMEK5 DD expression in transgenic plants leads to activation of 44 and 48 kD MAPKs and causes a rapid cell death. To compare the cell death induced by the expression of AtMEK5 DD with the HR-cell death induced by avirulence pathogen infection, we analyzed the activation of downstream MAP Kinase and induction of PR genes expression in permanent transgenic Arabidopsis plants. In-gel kinase activity assay revealed that the infection of Pseudomonas syringae DC3000 harboring Avr Rpt2 gene also lead to activation of 44 and 48 kD MAPKs. PAL, PR1 and PR5 were strongly induced in plants undergo- ing HR-cell death caused by the infection of P. syringae DC3000, while only the expression of PR5 was strongly in- duced in transgenic plants expressing AtMEK5 DD protein. NahG protein in AtMEK5 ×NahG plants cannot suppress DD the cell death induced by AtMEK5 . And AtMEK5 DD DD pro- tein expressed AtMEK5 ×NahG plants showed no signifi- DD cant change in salicylic acid (SA) level. All these suggest that the cell death induced by the activation of AtMEK5 is sali- cylic acid-independent. | LIUHongxia WANGYing ZHOUTianhong SUNYujing LIUGuoqin RENDongtao | 2004 | Chinese Science Bulletin2004，49，24： | 0 |
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