Transcriptome Analysis Of Populus Tomentosa Under Low Nitrogen Stress,cloning And Transformation Of MiR171c

miRNA is a class of non-coding small RNAs widely present in eukaryotes.It regulates the expression of target genes by cleaving m RNA or inhibiting translation of targets,and plays an important role in plant growth and development,responding to stress etc.The investigation of the expression changes of target genes in poplar under low nitrogen stress,identification of miRNA related to low nitrogen stress,and elucidation of its regulatory function in poplar could provide important genes for poplar breeding of nutrient use efficiency and lay a certain theoretical basis for the molecular regulation mechanism of trees in response to low nitrogen stress.In this investigation,Illumina sequencing was used to perform transcriptome analysis on tissue culture plantlets of Populus tomentosa under low nitrogen stress to identify profiles of gene expression at transcriptome level.Based on the gene expression data,the target genes of miRNAs related to low nitrogen stress of P.tomentosa were predicted,and the expression patterns of target genes were analyzed combined with q RT-PCR experiment.Furthermore,the promoter sequence of P.tomentosa pre-miR171c was predicted and successfully cloned,and its regulatory element composition was analyzed.The miR171c gene of P.tomentosa was thereafter cloned and the miR171c overexpression vector was constructed.The genetic transformation experiment of miR171c was initially carried out in 84 K.The main results of this research are given as follows:(1)Transcriptome sequencing of P.tomentosa treated with low nitrogen stress was conducted,and 104,843,476 clean reads were finally obtained.Based on the obtained clean reads,the two sample contigs were further assembled into 17,801(DN)and 82,908unigenes(KK),and a total of 59,152 unigenes were annotated.A total of 1,561down-regulated and 1,101 up-regulated differentially expressed genes(DEGs)were identified.There were 20 DEGs involved in the nitrogen metabolism pathway,and four pathways related to carbohydrate metabolism were also enriched.(2)miRNA sequences of poplar were further used as probes to scan gene sequences of transcritome data of P.tomentosa under low nitrogen stress.3,024 target genes corresponding to 242 miRNA members belonging to 131 miRNA families were obtained.There were 57 target genes that changed significantly after low nitrogen stress treatment,including 29 up-regulated target genes and 28 down-regulated target genes.miR393 acts on the target gene KAT2,which is involved in adjusting Na~+and K~+homeostasis.Target gene PIF3 of miR399 encodes the photosensitizer interaction factor PIFs protein.Target gene of miR171c encodes the DELLA protein of the GRAS family,which is involved in the resistance of plants to stress.These miRNAs and target genes may be related to poplar response to low nitrogen stress.(3)The promoter region of 1,631 bp upstream of miR171c of Populus tomentosa was cloned and sequenced,and its regulatory element composition was analyzed.The promoter was found to contain basic promoter elements including TATA-box and CAAT-box.Cis-acting elements were also found,including MYBCORE,WRKY71OS,ARR1AT related to the resistance of plants to stress;DPBFCOREDCDC3,PYRIMIDINEBOXOSRAMY1A related to hormone regulation,and IBOXCORE and SORLIP1AT related to light regulation,The miR171c gene may play a role through these cis-acting elements in the response of poplar to low nitrogen stress.(4)The miR171c of P.tomentosa with a length of 370 bp was cloned and the miR171c overexpression vector was constructed to engineering agrobacterium for gene transformation.The genetic transformation experiment was performed in P.tomentosa,and 5 miR171c overexpression plants were identified.This investigation should provide some theoretical clues for further exploring the function of poplar miRNA in response to low nitrogen stress,which is helpful to reveal the miRNA regulation mechanism of poplar in response to low nitrogen stress,and provide candidate genes for the molecular improvement of tree breeding of high nutrition use efficiency.