| The actins are important in eukaryotic organisms, which were essential for actin cytoskeleton, and involved in many processes during eukaryotic organisms development, Once the dynamic behavior of the actin cytoskeleton was destroyed, the plant will show morphological abnormalities more or less. With the cells, a variety of actin binding proteins (ABPs) are composed to maintain the actin dynamics with their functional diversity.As an important kind of actin binding proteins, LIM proteins participate in the process of actin assembly and disassembly in plants as reported, containing F-actin nucleation, severing, and capping activities. In spite of the regulation of LIM gene have researched in cotton fiber development, few reported the function of LIM gene in cotton pollen grow process. Our research has focused on the biochemstry function of two LIM gene (named GhPLIM1and GhPLIM2) highly expressed in cotton anthor, the works as follow:1、Subcelluar localization of GhPLIM1and GhPLIM2proteinsTo investigate the function of GhPLIM1and GhPLIM2protein in cotton cell, and explore whether the GhPLIM1and GhPLIM2can interaction with actin cytoskeleton as same as other LIM protein reported before, we build the pBI121-GhPLIM1-GFP and pMD-GhPLIM2-GFP two expression vectors, through agrobacterium-mediated transformation of LBA4404, the GFP fluorescence were be observed in the transgenic cotton callus cells, further observation under confocal microscope, we discovered that the GhPLIM1not only has cytoplasm localization but alsolocalized in the nucleus. In the cytoplasm, GhPLIM1-GFP formed a network, and the signal also accumulated in the cell nucleus. The subcellular localization of GhPLIM2observations is similar to GhPLIM1, so we conclude that GhPLIM1and GhPLIM2are both F-actin binding proteins, and have nuclear localization.2、GhPLIM1and GhPLIM2proteins interact with F-actinWe constructed two prokaryotic expression vectors, one is pET-28a-GhPLIM1, the other is pET-28a-GhPLIM2, then the His-tagged GhPLIM1and GhPLIM2protein were expressed in BL21, and purified on a nickel-nitrilotriacetic acid agarose(Ni-NTA) matrix, the High-speed cosedimentation assay indicated that both of GhPLIM1and GhPLIM2proteins can interact with F-actin, and formed the large relative molecular mass compounds, then sedimentation with the high-speed centrifugal force. 3、GhPLIMl and GhPLIM2proteins bundle act in filamentsThe concentreated GhPLIM1/GhPLIM2incubated with preformed F-actin, then the low-speed cosedimentation assay indicated that each of F-actin, GhPLIM1, GhPLIM2exist alone, it can not be settled with the low-speed centrifugal force. Once the preformed F-actin treated with GhPLIM1or GhPLIM2, either of them can be settled in the bottom with the low-speed centrifugal force. It indicated that GhPLIMl or GhPLIM2protein can bundle actin filaments,which increase the relative molecular mass in the low-speed centrifugal force.4、GhPLIMl stabilizes F-actinVarying concenteations of GhPLIM1incubated with preformed F-actin, then treated with Lat B (A kind of F-acin inhibitor, can make the depolymerization of actin filaments), the supernatants and pellets were separated by high-speed centrifugation. The result indicated that most F-actin accumulated in the pellet without treated with Lat B, by contrast, the addition of Lat B made part of F-actin depolymerize and was detected in the supernatant. When added GhPLIM1protein, it can protect F-actin from depolymerization in the presence of Lat B, and as GhPLIMl protein concentration increased, the function of stabilization is more obvious.5、Cotton transformation and identification of Gh WLIM5transgenic plantsApart from the above two LIM gene, we also studied the cotton GhWLIM5gene. Build pBI-35S-GhWLIM5RNAi、PBI-pTua9-GhWLIM5RNAi、pBI-PTua9-GhWLIM5OE (over expressing) three vetors, obtained a lot of genetically modified cottons through agrobacterium-mediated transformation of LBA4404(TO). At present, the transgenic material has received several line seeds (T1). Further analysis is under way. |
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