Study On Subcellular Localization And Biochemical Function Of Three Cotton LIM Proteins

Cotton is important economic crop in the world because the yield and quality of cotton fibre is associated with people’s daily lives. As known to all, there are many people in our country, so it is important to deal with people’s every wear. The quality of cotton fibre is also important for textile industry. High quality cotton fibre improves not only work efficiency but also the quality of products.LIM proteins are rich in cysteines and their molecular structures contain one or more LIM domains. The LIM domain (CX2CX17-19HX2CX2CX16-20CX2C/H/D for short) usually contains two zinc finger structures. The studies showed that plant LIM proteins were characterized by two kinds of functions. One kind of function is that some plant LIM proteins work as a target genes’ regulator and the other function is about regulating actin cytoskeleton. By far, there are some studies on LIM proteins. However, LIM proteins’function in cotton fiber is not clear. We isolated and characterized three cotton LIM genes and they were designated as GhLIM2, GhLIM5and GhLIM6. Our work showed that three cotton LIM genes were predominantly expressed in fibre tissue, indicating they could play significant roles in fibre cells elongation. To study the functions of three LIM proteins, we worked from the following areas:the localization of LIM proteins, the interaction between LIM proteins and F-actin and the regulation to cytoskeleton. The detailed results are as followed:1. The localization of three GhLIM in the cellsAt first, we constructed the vector of PBI121-GhLIM2/5/6-GFP. Then the vector was transformed in cotton cells, Arabidopsis thaliana cells and tobacco’s leaf cells by mediation of agrobacterium. The observation under the confocal microscopy showed GhLIM5-GFP fusion proteins were evidently diffuse in cotton and Arabidopsis thaliana cell nuclei and clearly displayed the network distributions in cytoplasm. The same results of GhLIM6-GFP fusion proteins were observed in cotton and tobacco leaf cells. To further confirm the localizations of GhLIM5/6-GFP fusion proteins, we used rhodamine-phalloidin and confirmed the fusion proteins located on the cell cytoskeleton2. The recombinant GhLIM proteins obtained from prokaryotic expression system directly bind to F-actin We obtain soluble GhLIM proteins from prokaryotic expression system. Then the high-speed cosedimentation assay was employed to confirm the interaction between GhLIM proteins and F-actin. The result showed that GhLIM proteins could directly bind to F-actin.3. The recombinant GhLIM proteins obtained from prokaryotic expression system bundle the F-actinThe soluble GhLIM proteins were obtained from prokaryotic expression system. Then the low-speed cosedimentation assay was employed to confirm whether GhLIM proteins bundle F-actin. The result showed that GhLIM proteins could bundle F-actin.4. Suppression of GhLIM6expression by RNAi technology in cottonTo study the functions of GhLIM gene family in the cotton fibre development, we employed RNAi technology to suppress the GhLIM6expression. By far, we have obtained many transgenic cotton plants and related analysis is going on.