Cloning And Functional Preliminary Studies Of AmrMYBR1 And AmrR2R3MYB Genes From Agropyron Michnoi Roshev.

Agropyron michnoi Roshev.is a perennial rhizoid or rhizo-sparse herbaceous plant of Agropyron.It is a tetraploid（2n=28）,belonging to the grassland xerophyte group,mainly growing in grassland and desert steppe areas.MYB transcription factors play an important role in the regulation of plant stress responses.In this study,MYB transcription factor genes were isolated from Agropyron michnoi Roshev.by homologous sequence cloning,and their expression characteristics and functions were analyzed.This study laid a foundation for the mining and utilization of resistance genes in Agropyron michnoi.The results are as follows:1 For the first time,a MYB transcription factor gene with 1R structure was cloned from A.Michnoi Roshev.,which was named AmrMYBR1,with a maximum open reading frame of 783 bp,encoding 260 amino acids.The molecular weight of the deduced protein was 28.59 KD and the isoelectric point was 7.78.A R2R3MYB transcription factor gene named AmrR2R3MYB was also cloned from the A.michnoi Roshev.The maximum open reading frame of AmrR2R3MYB was 969 bp,encoding 323 amino acids.The molecular weight of the deduced protein was 35.45 KD,and the isoelectric point was 5.58.Both two genes were hydrophilic and unstable proteins.Protein sequence alignment and phylogenetic tree analysis showed that AmrR2R3MYB gene was highly conserved,and both genes were closely related to MYB protein in Triticum.2 qRT-PCR was used to analyze the expression pattern of the AmrR2R3MYB gene.The results showed that the expression level of AmrR2R3MYB was highest in leaves,followed by stems and roots,and lowest in spikes.The expression of AmrR2R3MYB was positively regulated by drought stress and ABA,but inhibited by salt stress and GA.3 GUS activity in transfected Arabidopsis thaliana protoplasts showed that AmrR2R3MYB had self-activation function and up-regulated stress-responsive genes such as ERD10,RD17,RD22,RD29B and COR15A.4 By constructing p CAMBIA1300-AmrR2R3MYB-c YFP vector and transiently transfecting tobacco leaves,AmrR2R3MYB was localized in the nucleus.5 The fusion expression vector p CAMBIA1302-AmrMYBR1 was constructed and transformed into rice protoplasts.The subcellular localization results showed that AmrMYB1 was localized in the nucleus and cytoplasm.p CAMBIA1302-AmrMYBR1 was transformed into rice callus.After screening,23 T₀transgenic rice plants and corresponding T₁transgenic rice seeds were obtained.These results provided a strong basis for the exploration and utilization of excellent genes in Agropyron michnoi Roshev.’Baiyinxile’and enriched the resistance genes in Agropyron.