Screening Of Proteins Interacting With PhCOG8 And Their Functional Identification In Petunia

The eukaryotic cell contains a variety of organelles.The cytoplasmic membrane and these membranous structures constitute a complex biofilm system.The exchange of a number of substances and information in the systems is the basis of life activities in cell's.Vesicular trafficking is a major form of transport of substances within eukaryotic cells.The vesicle transport process is divided into four basic steps.They are budding,translocation,thrombectomy,and fusion.The COG complex participates in the thrombectomy process.There are eight subunits in COG complex.They are associated with architecture of Golgi,intracellular trafficking and glycoprotein modifications.At present,the subunit function of the COG is already clear in mammals and yeast,but there is little related research in plants.In this study,we observed the phenotype of petunia 'violet' after silencing COG8.Then we constructed a subcellular localization vector.We obtained some proteins interacting with PhCOG8 by yeast two-hybrid system and analyzed the interaction between PhCOG8 and these proteins by BiFC.Finally,we identified the function of the interacting proteins and observed the phenotypes by the VIGS.Then we found the similarity between the PhCOG8 silenced plants and the interacting proteins silenced plants.The main results can be listed as follows.1.The growth points of PhCOG8-silenced plant are inhibited and the new leaves are malformed.PhCOG8 protein located in cytoplasm and cell membrane.2.By yeast yeast two-hybrid assays,there were eight proteins,PhVHA-d2,PhAt,PhVPE,PhMYB308,PhATGI8,PhSAM3,PhWrap53 and PhERD3,interacting with PhCOG8.One-to-one validation of yeast two-hybrid showed that there is an interaction between PhCOG8 and five proteins,PhVHA-d2,PhAt,PhVPE,PhMYB308 and PhATGI8.PhSAM3 was self-activated.However there was no interaction between PhWrap53 and PhCOG8 or PhERD3 and PhCOG8.3.Fluorescence was observed in PhAt,PhVPE and PhCOG8 in the BiFC experiment,whereas no fluorescence was observed in PhSAM3 and PhCOG8,PhVHA-d2 and PhCOG8,PhMYB308 and PhCOG8,PhATGI8 and PhCOG8.4.PhVHA-d2,PhAt,PhVPE,PhMYB308,PhATGI8 and PhSAM3 were silenced in petunia plants by the VIGS.Compared with the wild type plants,the early growth of the PhVHA-d2-silenced plants was inhibited,which is similar with that of PhCOG8 silencing.Compared with the wild type plants,the overall leaf shape of PhSAM3 silencing was narrow.