Verification And Application Of A Novel MiRNA Pathway Screening System In Arabidopsis Thaliana

MicroRNAs(miRNAs)are 20?24-nucleotide(nt)non-coding RNAs that play key regulatory roles in developmental and physiological processes in plants.miRNA are encoded by MIR gene.MIR genes are transcribed into primary-miRNA transcripts with stem-loop structure by RNA polymerase II(Pol II).The pri-miRNA is then processed by Dicer like 1(DCL1)into the pre-miRNA,which is further processed into the miRNA/miRNA* duplex.The duplex is methylated by HUA ENHANCER1(HEN1)and selected to recruited to the RNA-induced silencing complex(RISC)whichcontains specific miRNAs,scans for complementary m RNA transcripts and directs the cleavage or translational repression at the target m RNAs.Thus miRNAs can regulate the growth and development of the plants through regulating the expression of their target genes.In order to screen the new components that are involved in miRNA biogenesis,turnover and movement,we established a forward genetic screening system by using a Arabidopsis thaliana transgenic line(SUC2:ami R-SUL).In the SUC2: ami R-SUL transgenic Arabidopsis,an artificial miRNA is expressed under the phloem-companion cell specific promoter SUCROSE-PROTON SYMPORTER 2(SUC2).This triggers non–cell-autonomous RNAi of the ubiquitously expressed m RNA of SULFUR(SUL)homolog CH42,which is involved in the biogenesis of chlorophyll,generating a leaf chlorotic phenotype that expands 10 to 15 cells beyond the vasculature.After EMS mutagenesis on the transgenic line,and screening on the M2 plants,one stabel mutant line(SUP-E45)was isolated for which the true leaf veins significantly turn green.The results shows that the altered phenotype of SUP-E45 mutant was caused by a point mutation in the gene coding for the AGO1 protein.AGO1 protein is crucial in the miRNA pathways,which recruits mature miRNA to form mi RISC silence complex(micro RNAs-Induced Silencing complex,mi RISC)to negatively regulate the expression of target genes.Another mutation line,ab58,was isolated for which the true leaf veins significantly turn green.ab58 mutants exhibited very distinct phenotypes with normal number of visible leaves,but smaller leaves in size and curly in shape.Besides,ab58 is sensitive to temperature.The data indicated that the mutanted gene was BON1,a member of copine gene family.In this mutant the endogenous miRNAs levels were decreased.The results indicated that BON1 may be a new component involving in miRNA pathway.More studies will be carried out to investigate the functions and molecular mechansim of BON1 in miRNA pathway in future.This study not only verified the feasibility of SUC2: ami R-SUL Arabidopsis system,but also identified a putative new component of miRNA pathway.All these study will provide a fundamental basis for the miRNA pathway studies.