The Functional Analysis Of AAaMYB16 Transcription Factor Responses To Freezing Tolerance In Actinidia Arguta

Actinidia arguta is a common wild fruit tree in northern China,which has high economic value.Due to the influence of latitude,low temperature stress occurs frequently in northern China,which has a negative impact on plant growth and development,yield and quality.In this experiment,the AaMYB16 gene of Actinidia arguta was cloned.The plant expression vector GV1300-AaMYB16 was constructed to transiently transform the isolated leaves of Actinidia arguta and stably transform Arabidopsis thaliana,to analyze the cold-resistance function of R2R3 MYB transcription factors by the method of molecular biology,plant physiology,biochemistry and genetics.The main results of the experiment are as follows:1.The full length of AaMYB16 of Actinidia arguta is 1200 bp,encoding 399 amino acids.As a member of R2R3-MYB family,phylogenetic tree showed that the protein was closely related with homologous proteins from Actinidia rufa,Actinidia deliciosa and Vitis vinifera L.2.The plant expression vector GV1300-AaMYB16 was constructed to constructed to transiently infect onion epidermal leaves and tobacco leaves for subcellular localization.The result showed that AaMYB16: GFP was located in nucleus.Under freezing treatment,the accumulation of ROS in isolated transgenic Actinidia arguta leaves was higher than that of the control.The relative expression of low temperature stress related genes Aa CBF,Aa Gol S,Aa KIN1,Aa SAG21 and Aa COR47 were lower than that in the control.The results of stable transformation of Arabidopsis thaliana showed that the survival rate,leaf water content,POD,CAT,APX,SOD enzyme activities,proline content,chlorophyll content and anthocyanin content of transgenic Arabidopsis thaliana were lower than those of the control under freezing stress,The wilting rate,ion leakage,ROS accumulation and MDA content were higher than those in the control.The relative expression of genes related to chilling stress At CBF1,At CBF2,At CBF3,At KIN1 and At COR47 were lower than those in the control.This result showed that overexpression of AaMYB16 lead to cold sensitivity of isolated Actinidia arguta leaves and Arabidopsis thaliana.3.The yeast expression vector pGBKT7-AaMYB16,the bimolecular fluorescence complementary expression vector p SPYNE-AaMYB16 and the double luciferase expression vector p CAMIA1300-n LUC-AaMYB16 were constructed to verify the interaction between AaMYB16 and bHLH protein by yeast two hybrid,bimolecular fluorescence complementarity and double luciferase.The experiment results showed that AaMYB16 protein had selfactivating activity.There was no interaction between AaMYB16 protein and AabHLH18 protein and AaMYB16 protein has the interaction with AabHLH137 protein.