Functional Analysis Of LncRNA167 And Its Cleavage Product MiR167c In Saline Stress Of Medicago Truncatula

Alfalfa(Medicago sativa L.)is an important leguminous forage.In actual production,soil salinization seriously restricts the yield of alfalfa.The research on the molecular mechanism of salt tolerance regulation of alfalfa will provide theoretical basis for the breeding of salt tolerant varieties.Although the feeding value is low,Medicago truncatula is often used as a model plant to study the molecular regulation mechanism of alfalfa due to its small diploid genome,easy transformation and high similarity with alfalfa genome.In recent years,more and more studies have shown that non-coding RNAs such as lncRNAs(long non-coding RNAs)and miRNAs(micro RNAs)play an important role in the regulation of abiotic stress in plants.In this study,a salt-responsive lncRNA(Mt-lncRNA167)and its cleavage product,MT-miR167 c,were preliminarily studied from Medicago truncatula.(1)The new members Mt-lncRNA167 and Mt-miR167 c were identified in Medicago truncatula.In the early stage,deep high-throughput sequencing of lncRNAs was conducted on R108,and a large number of lncRNAs responding to salt stress were screened.A novel lncRNA was identified,and it was predicted that the lncRNA could splice to form a miRNA.Sequence analysis showed that the structure of its precursor matched the secondary structure characteristics of miRNA,belonging to the miR167 family,and the miRNA was named Mt-miR167 c and the lncRNA was named Mt-lncRNA167.(2)The expression pattern of Mt-lncRNA167 was similar to that of Mt-miR167 c,and the regulatory network of lncRNA167-miR167c-ARF6/8 was preliminarily verified from the expression level.Tissue differential expression analysis confirmed that the expression pattern of Mt-miR167 c was similar to that of Mt-lncRNA167,while that of target genes MtARF6/8 was contrary.The expression levels of miR167 c and target gene MtARF6/8 were opposite under salt and drought stress.The results indicated that MtlncRNA167 and Mt-miR167 c responded to various abiotic stresses,and the interaction network of lncRNA167-miR167c-ARF6/8 was preliminarily verified.(3)Mt-lncRNA167 and Mt-miR167 c play important roles in growth development and abiotic stress regulation.Overexpression of Mt-lncRNA167 and Mt-miR167 c in Arabidopsis thaliana showed a fertility defect phenotype.This suggests that Mt-miR167 c,like other members of the family,plays an important regulatory role in plant fertility.Overexpression of Mt-lncRNA167 and Mt-miR167 c enhanced the resistance of Arabidopsis thaliana to salt stress at germination and seedling stage,but decreased the resistance to drought stress at seedling stage.The overexpression of Mt-lncRNA167 and Mt-miR167 c significantly inhibited the growth and development of Medicago truncatula,mainly including low seed setting rate,dwarfed plant,increased branchings and smaller seeds.(4)The role of Mt-lncRNA167 as a precursor of Mt-miR167 c was preliminarily confirmed.In this experiment,the overexpressed plants of Mt-lncRNA167 and the overexpressed plants of Mt-miR167 c had the same phenotype.The expression level analysis of all the lines showed that there was an interaction network relationship between Mt-lncRNA167 and Mt-miR167 c as well as target gene MtARF6/8.Therefore,we believe that Mt-lncRNA167 plays a role as a precursor of miR167 c,and Mt-miR167 c affects the growth and development of plants by inhibiting the expression of ARF6/8.In conclusion,a novel lncRNA,Mt-lncRNA167,and its cleavage product,Mt-miR167 c,were identified and analyzed in this study.Functional analysis showed that Mt-lncRNA167 and Mt-miR167 c played an important role in the regulation of growth and development and abiotic stress.This study provided a theoretical basis for the future genetic improvement of alfalfa.