Cloning And Functional Research Of Novel MiRNAs In Populus Szechuanica Upon Infection With Melampsora Larici-populina

Poplar is one of the most important economic and ecological trees in the world,and it is also a model tree for forest tree genetic research.Leaf rust caused by Melampsora laricipopulina is an important leaf disease in the seedling and sapling stages of poplar,which often causes great losses in poplar production.MicroRNA?miRNA?is a kind of small-molecule single-stranded non-coding RNA of 20²5 nt.In plant,miRNA participate plant growth,development and responses to stresses through repressing the target gene expression by generally direct cleavage of complementary mRNA.Studying miRNAs can help reveal the molecular mechanisms of many life phenomena,and which will help to provide insights into the genetic improvement of crops and trees.In this paper,two novel miRNAs related to rust resistance predicted by high-throughput sequencing were detected,their target genes were subjected to cleavage validation,full-length target genes were amplified,and functions of target genes were predicted.Finally,the overexpression vector of novel miRNA precursor gene was constructed in order to be transferred into poplar by Agrobacterium tumefaciens mediated method,so as to further understand the function of miRNA when the poplar is infected by rust fungus.The specific research results are as follows:1.Detection of novel miRNAs from Populus szechuanica leaves infected with M.laricipopulinaIn the early research of our laboratory,the sRNA library was constructed from P.szechuanica leaves during compatible and incompatible interations with M.larici-populina,In this experiment,novel_mir₁1 and novel_mir₃57 were selected from these novel miRNAs for detection.The stem-loop primer was designed by stem-loop method,and the DNA band corresponding to the target size was detected,which confirmed the existence of novel_mir₁1 and novel_mir₃57 in Populus szechuanica.2.Validation of novel miRNA target genesThe novel_mir₁1 and novel_mir₃57 target genes and cleavage sites were validated by RLM-5'RACE,and two?Potri.014G009300.1 and Potri.014 G 002000.1?of the predicted target genes of novel_mir₁1 and three?Potri.T044800.1,Potri.T025800.1 and Potri.018G135600.1?of the predicted target genes of novel_mir₃57 were validated.It was found that novel_mir₁1 and novel_mir₃57 are highly complementary to their target genes respectively,and both cleave the target gene.At the same time,the location of these cleavage sites was not fixed,some were located within the complementary region of the miRNA and the target gene,and some were located outside the complementary region,but most of the miRNAs were cleaved in the complementary region.3.Full-length amplification and novel miRNA regulation of target genesFrom the validated target genes,one target gene of novel_mir₁1 and novel_mir₃57 was selected respectively for full-length amplification and bioinformatics analysis.The target gene of novel_mir₁1 was 3899 bp in length and encoded 942 amino acids,including a 2829 bp open reading frame.The target gene of novel_mir₃57 was 6010 bp in length and encoded 621 amino acids,including a 1866 bp open reading frame.Conservative domain analysis of predicted proteins of target genes revealed that both sequences have typical NB-ARC and LRR domains with disease resistance gene characteristics,and the target genes of novel_mir₁1 and novel_mir₃57 were named PsRPM1 and PsRPS2,respectively.The amion acid sequence alignment of the deduced proteins encoded by these two target gene using BLASTP in NCBI revealed that the deduced proteins were the most similar to the NBS-LRR disease resistant proteins of Populus trichocarpa.Therefore,PsRPM1 and PsRPS2 were presumed to be an NBS-LRR-like disease-resistant protein gene.qRT-PCR was used to detect the temporal dynamic expression of miRNA and its disease-resistant target genes in P.szechuanica leaves infected with both virulent and avirulent isolates of M.larici-populina in different time periods after inoculation.The results showed that novel miRNA negatively regulates the expression of target genes in affinity and non-affinity.There are differences in the interaction.4.Construction of pze-miR11 expression vectorThe precursor sequence containing novel_mir₁1 was cloned from the P.szechuanica,and ligated with the vector pBI121 to construct a plant overexpression vector of pre-pze-miR11,which was successfully transferred into Agrobacterium tumefaciens GV3101 for further validating the pze-miR11 regulation of disease resiatence genes through genetic transformation of poplar.