[Objective] The aim was to investigate the dimer formation between the movement proteins（MP）in barely yellow dwarf virus by using the technology of bimolecular fluorescence complementation technology and to further ...[Objective] The aim was to investigate the dimer formation between the movement proteins（MP）in barely yellow dwarf virus by using the technology of bimolecular fluorescence complementation technology and to further study the relationship between MP homodimerization and viral movement.[Method] The DNA sequence of bimolecular fluorescent complementary vector containing cloning multiple cloning sites,35S promoter and terminator was cloned into the expression vector pCAMBIA1300,which replicates at a higher copy number in E.coli.Then,the BYDV-MP gene fragment was amplified in the presence of the whole BYDV-PAV cDNA sequence as template and the primers designed according to the BYDV-MP gene sequence from GenBank,cloned into the modified bimolecular fluorescent complementary vectors pCAMBIA1300-NE and pCAMBIA1300-CE.The resulting vectors were transformed into Agrobacterium by electroporation method and infiltrated into the tobacco leaf.Protein interactions were observed under fluorescence microscope.[Result] Yellow fluorescence could be viewed in the leaves co-infiltrated with Agrobacterium carrying pCAMBIA1300NE-MP and pCAMBIA1300CE-MP at 2-5 d post-infiltration,while yellow fluorescence could not be observed in negative control groups.[Conclusion] BYDV-MP formed homodimers in plant cells.The results can provide theoretical basis for further in-depth research about the movement process and mechanism of BYDV.展开更多
Transforming growth factor-β （TGF-β） binds with two transmembrane serine/threonine kinase receptors, type Ⅱ （TβRII） and type Ⅰ receptors （TβRⅠ）, and one accessory receptor, type Ⅲ receptor （TβRⅢ）, to...Transforming growth factor-β （TGF-β） binds with two transmembrane serine/threonine kinase receptors, type Ⅱ （TβRII） and type Ⅰ receptors （TβRⅠ）, and one accessory receptor, type Ⅲ receptor （TβRⅢ）, to transduce signals across cell membranes. Previous biochemical studies suggested that TβRI and TβRIII are preexisted homo-dimers. Using single-molecule microscopy to image green fluorescent protein-labeled membrane proteins, for the first time we have demonstrated that TβRI and TβRⅢ could exist as monomers at a low expression level. Upon TGF-β1 stimu- lation, TβRI follows the general ligand-induced receptor dimerization model for activation, but this process is TβRⅡ- dependent. The monomeric status of the non-kinase receptor TβRⅢ is unchanged in the presence of TGF-β1. With the increase of receptor expression, both TβRI and TβRIII can be assembled into dimers on cell surfaces.展开更多
基金Supported by National Natural Science Foundation of China(30870109)~~
文摘[Objective] The aim was to investigate the dimer formation between the movement proteins（MP）in barely yellow dwarf virus by using the technology of bimolecular fluorescence complementation technology and to further study the relationship between MP homodimerization and viral movement.[Method] The DNA sequence of bimolecular fluorescent complementary vector containing cloning multiple cloning sites,35S promoter and terminator was cloned into the expression vector pCAMBIA1300,which replicates at a higher copy number in E.coli.Then,the BYDV-MP gene fragment was amplified in the presence of the whole BYDV-PAV cDNA sequence as template and the primers designed according to the BYDV-MP gene sequence from GenBank,cloned into the modified bimolecular fluorescent complementary vectors pCAMBIA1300-NE and pCAMBIA1300-CE.The resulting vectors were transformed into Agrobacterium by electroporation method and infiltrated into the tobacco leaf.Protein interactions were observed under fluorescence microscope.[Result] Yellow fluorescence could be viewed in the leaves co-infiltrated with Agrobacterium carrying pCAMBIA1300NE-MP and pCAMBIA1300CE-MP at 2-5 d post-infiltration,while yellow fluorescence could not be observed in negative control groups.[Conclusion] BYDV-MP formed homodimers in plant cells.The results can provide theoretical basis for further in-depth research about the movement process and mechanism of BYDV.
基金This work was supported by the National Natural Science Foundation of China （90713024, 20821003, 30921004）, the National Basic Research Program of China （2007CB935601, 2010CB833706） and the Chinese Academy of Sciences.
文摘Transforming growth factor-β （TGF-β） binds with two transmembrane serine/threonine kinase receptors, type Ⅱ （TβRII） and type Ⅰ receptors （TβRⅠ）, and one accessory receptor, type Ⅲ receptor （TβRⅢ）, to transduce signals across cell membranes. Previous biochemical studies suggested that TβRI and TβRIII are preexisted homo-dimers. Using single-molecule microscopy to image green fluorescent protein-labeled membrane proteins, for the first time we have demonstrated that TβRI and TβRⅢ could exist as monomers at a low expression level. Upon TGF-β1 stimu- lation, TβRI follows the general ligand-induced receptor dimerization model for activation, but this process is TβRⅡ- dependent. The monomeric status of the non-kinase receptor TβRⅢ is unchanged in the presence of TGF-β1. With the increase of receptor expression, both TβRI and TβRIII can be assembled into dimers on cell surfaces.
