Regulation Mechanism Of Populus AS1a Gene In Secondary Growth Process

AS1 is a R2R3-MYB transcription factor that regulates the establishment of proximal to distal polarity in arabidopsis leaves,but its function in poplars is unknown.In the RNA-seq data obtained in the previous study,it was found that the expression level of arabidopsis AS1 homologous gene in phloem was significantly higher than that in xylem（named AS1a）.In this paper,AS1 a gene was used as the research object.The function and molecular mechanism of AS1 a in populus secondary growth and development will be further studied by constructing transgenic plants with overexpression and dominant inhibition of AS1 a gene,section analysis,tobacco and double lucifase transient,yeast double hybridization,yeast single hybridization,RNA-seq,DAP-seq and other experiments.The main research results available are as follows:（1）The full-length CDS sequence of AS1 a was cloned using 84 K poplar CDS as template.Compared with the genome of P.tomentosa,the base similarity and amino acid similarity of AS1 a were 97.05% and 95%,respectively.Overexpressed AS1 a plant transformation vector 35S::AS1a and dominant inhibitory plant transformation vector 35S::AS1a::SRDX were constructed,and 11 and 9 independent transgenic lines 35S::AS1a and35S::AS1a::SRDX were obtained by agrobacterium-mediated leaf plate infection and transformation,respectively.（2）Transient tobacco transformation assay demonstrated that AS1 a protein was localized to the nucleus.（3）AS1a overexpressed plants showed no significant differences in plant height,stem diameter and internode number compared with wild type.The results showed that the development of xylem was significantly advanced and the width of xylem was significantly increased.The phenotype of dominant inhibited plants was similar to that of overexpressed plants.The number of adventitious roots in transgenic plants increased,the number and length of lateral roots on each adventitious root decreased,and the surface area and volume of each adventitious root decreased.The length,width and petiole length of transgenic plants were larger than those of wild type.（4）Transcriptional activity assay proved that AS1 a was a transcription inhibitor.（5）Transcriptome sequencing（RNA-seq）was performed on 11# lines of AS1 a overexpressed plants.The results showed that there were 2965 differentially expressed genes,of which 1198 genes were significantly up-regulated and 1767 genes were significantly down-regulated.GO analysis of the differential genes showed that the genes of cellulose biosynthesis,lignin biosynthesis,secondary metabolism and secondary metabolite synthesis were significantly up-regulated,while the genes of lignin decomposition and phenylpropane metabolism were significantly down-regulated.（6）Yeast double hybrid experiment showed that AS1 a interacted with AS2.（7）AS1a can bind to the promoter regions of Pag PIN5 b and Pag KNAT2/6b in yeast monozygotic experiments.