Identifiication Of Interacting Proteins Of Cytokinin Transporter AtABCG14

Cytokinin is a phytohormone that has important function in plant growth and development,disease resistance,stress resistance,absorption and utilization of nutrients and leaf senescence.The research about synthesis metabolism and signal transduction of cytokinin has been studied for a long time,but study of the molecular mechanisms of cytokinin transport is still unclear.AtABCG14 is the first discovered protein related to long-distance transport of cytokinin.AtABCG14 is mainly responsible for the transport of root-synthesized cytokinin to the ground tissue through the xylem to promote the growth and development of the ground tissue.AtABCG14 is a semi molecular transporter of the family of ABC transporters,so it need to interact with other protein to complete cytokinin transport.But until now the protein interacting of ABCGA14 has not been reported.The purpose of this study is to identify the interaction protein of AtABCG14,and illustrate the transport mechanism of cytokinin.First we obtained the interacting proteins of AtABCG 14 using the co-immunoprecipitation method and liquid chromatography-mass spectrometer(LC-MS/MS),then we identified proteins interacting with ABCGA14 by the molecular biology,genetics and other research methods.The concrete results are as following:(1)We obtained 124 interacting proteins of AtABCG14 using the co-immunoprecipitation method and liquid chromatography-mass spectrometer,and then we selected 26 possible proteins from the 124 proteins to do the subsequent experiment.(2)The candidate genes were constructed on the N-terminal vector of luciferase protein and the AtABCG14 gene was constructed on the C-terminal vector of luciferase protein,then two vectors were simultaneously transferred into to verify the interaction between candidate protein and AtABCG14 in tobacco.The results showed that there were 15 proteins interacting with AtABCG14 in tobacco.(3)The T-DNA insertion mutants of 10 candidate genes were identified,than the phenotype of the mutants were statistically analyzed.The results showed that ahal mutant and abcg36 mutant rosette leaves smaller,inflorescence reduced,stem shorter,these phenotypes are consistent with abg14 mutant,which are phenotypes absent from cytokines.In addition,in this study,ahal mutant and abcg36 mutant were treated with 6-BA,and the change of root length of mutants was statistically analyzed to detect the response of ahal mutants and abcg36 mutant to 6-BA.The results showed that ahal mutant is insensitive to 6-BA,which is similar to abcgl4 mutant,and abcg36 mutant is sensitive to 6-BA,which is similar to wide type.(4)The candidate genes of AHA1 and ABCG36 were built into the yeast expression vector pPR3N,and the AtABCG14 gene was constructed to the yeast expression vector pBT3N,then two vectors were transferred to the yeast strain NMY51 to verify their interaction.The results showed that AHA1 is interacting with AtABCG14 in yeast,while ABCG36 is not interacting with AtABCG 14 in yeast.(5)The expression of ARR5 gene in aha1 mutant and abcg36 mutant was detected by the GUS staining of ProARR5::GUS/ahal and ProARR5::GUS/abcg36 transgenic plant.GUS staining showed the level of cytokins in ahal mutant of the ground tissues is greatly reduced,and the level of cytokins in abcg36 mutant of the ground tissues is slightly reduced.