Identification Of Target Genes Of MiRNA In Poplar Infected With Lonsdalea Quercina And The Genetic Transformation Of MiR482c

The bacterial canker disease of Populus×euramericana is a stem disease caused by Lonsdalea quercina subsp.Populi,which seriously threatens the growth and survival of poplar.In order to elucidate the disease resistant related genes in poplar,and explore the molecular mechanism of poplar disease resistance,target genes of miRNA were identified from transcriptomic data of poplar‘zhonglin46’after L.quercina infection in this investigation.The promoter of miR482c which was responsive to the infection of pathogen was cloned and analyzed in poplar,and the genetic transformation of miR482c was initially carried out in Populus alba×P.glandulosa clone‘84K’.Meanwhile,based on the previous study in our research group,the interaction proteins of LAR,which has been found to be associated with poplar disease resistance and is an important enzyme in secondary metabolic pathways,were screened and verified.The results of this investigation are given as follows:（1）The miRNAs in poplar were used as probes to predict the target genes from the differentially expressed genes in poplar inoculated with or without L.quercina and the bioinformatics analysis of the obtained target genes was carried out.A total of 566 target genes corresponding to 276 miRNAs belonging to 127 miRNA families in poplar were screened,involved in multiple pathways such as plant pathogen interaction,plant hormone signaling transduction,and phenylpropanoid biosynthesis.Among them,miR482,miR6459,miR7812,miR7835,and miR396 target RPS2,NBS-LRR,AUX1,CCR,and Ca²⁺transmembrane transporter genes respectively,were involved in the regulation of poplar response to pathogen infection.Target gene prediction results further showed that miR482 family targeting 23differentially expressed genes,was the miRNA family with the largest number of targets in this investigation.Four of the differentially expressed genes targeted by miR7835 encode CCR enzymes in the phenylpropanoid biosynthetic pathway,which might participate in the disease resistant through affecting the synthesis of lignin in poplar.（2）In this study,the promoter of miR482c was successfully cloned in P.tomentosa.The analysis results of promoter sequence showed that a large number of disease resistance-related cis-acting elements exists in the upstream sequences,including MYB,MYC and WRKY transcription factor binding sites,hormone signal response elements ERE and CGTCA-motif,and disease resistance related elements GT1-motif and SEBF.（3）The phylogenetic tree of the miR482 family was constructed by neighbor joining method.The miR482 family was showed to be closely related with the Solanaceae and Malvaceae plants.The pCAMBIA2300-35S-OCS::miR482c overexpression vector and pCAMBIA2300-35S-OCS::STTM482c target silencing vector were constructed.After genetic transformation of Populus alba×P.glandulosa clone‘84K’mediated by using agrobacterium,three transgenic plants with miR482c overexpression were initially obtained.（4）Based on the yeast two-hybrid system,12 genes were initially screened from the Populus alba×P.glandulosa clone‘84K’cDNA library using the mating method,and the corresponding proteins could interact with LAR.Furthermore,plastocyanin,ABC transporter,and ubiquitin protein were validated to interact with LAR validation through yeast two-hybrid experiment.The above results should provide candidate genes for poplar breeding of disease resistance,and clues for the elucidation of the mechanism of resistance regulation by miRNAs in trees,and the construction of LAR centered regulatory network in response to pathogen infection.