Characterization Of Stress-Responsive Bna-miR169 And Its Target Genes BnNF-YA In Canola (Brassica Nap Us)

MicroRNAs are a class of endogenous non-coding RNA whose base number is 20-22bp.The primary miRNAs(pri-miRNAs)transcript was from the miRNA genes,and then processing to the mature microRNAs.Mature miRNA can combine with the AGO protein to form a silencing complex RISC(RNA-induced silencing complex).RISC bind to the cleavage site of a target gene by complementary base pairing,and then cut or inhibit the mRNA of target gene.Previous studies have showed that plant miRNA plays an important role in many physiological processes in plants,such as growth,dormancy and stress response.Some of the plant miR169 members and its target NF-YAs have been reported to be involved in the drought stress responses of plants.In this study,we chose canola cultivarwhich named 'Nanyanyou 1'(Brassica napus)as the experimental material.The sequence of mature miRNA and pri-miRNA were gotten from the miRBase database by blasting.Through the analysis of the expression abundance in the degradation sequencing database,we find five genes with high expression abundance.Then through the analysis of degradated sequence in the database and online target gene prediction,we finally chose bna-miR169m,bna-miR169n and bna-miR169o(new family member)as well as their potential target genes BnNF-YA3,BnNF-YA9,BnNF-YA2/12.Mature sequence of bna-miR169o is blasted in the cabbage genome database(BRAD),and there are three match sites in the cabbage genome.We downloaded the sequence which contained the both side of the corresponding region of 200bp,and designed primers for PCR amplification.Finally,we successfully obtained the sequence of bna-miR169o in the chromosome A08.Through analysis of online DNA folding prediction,bna-miR169o was determined to have the typical structure of miRNA genes.Through analysis of expression patterns for bna-miR169 gene family under abiotic stress(150mM NaCl,15%(w/v)PEG and 100?M ABA)by real-time quantitative PCR,experimental results showed that all members of bna-miR169 were induced in the three stress,but the expression pattern of each gene was different.By using canola genomic DNA as a template,promoter sequences of bna-miR169m/n/o were cloned.Using the online promoter regulatory element prediction database PLACE and plantCARE,we identified cis-acting elements related with stress in the promoter of three genes,indicating that expression of bna-miR169m/n/o may be regulated by abiotic stress.Using 5'RACE and transient expression in Nicotiana benthamiana,we verified that bna-miR169m/n/o can potentially cut their target genes BnNF-YA2/3/9/12.5'RACE has successfully proved that BnNF-YA2/3 were cutted at the predicted cleavage site,but BnNF-YA9/12 were not detected.The result of transient expression in Nicotiana benthamiana showed that bna-miR169m/n/o can potentially cut their target genes BnNF-YA2/3/9/12 at the cleavage site.The overexpression vector of bna-miR169m/n/o and BnNF-YA2/3/9/12 were constructed,and transformed to Agrobacterium EHA105 and finally transfered into the wild-type Arabidopsis thaliana(Col-0)by using dip method.We have got transgenic homozygote of bna-miR169 and BnNF-YA12,and F2-generation of the rest transgenic plants.The next step is further to screen the homozygotes.The result of physiological experiments for bna-miR169o and BnNF-YA12 transgenic homozygotes showed that overexpression of BnNF-YA12 plants were more sensitive to salt,drought and ABA treatment.Phenotype of overexpression of bna-miR169o plants and wild-type have no significant difference.Probable cause is a target gene of bna-miRl 69o does not exist in the Arabidopsis thaliana.