Cloning And Characterization Of Zma-miR169c And Its Target Gene ZmNF-YA13 From Zea Mays

Abiotic stress limits plant growth and productivity.Micro RNA has some effectson plant resistance to abiotic stresses.In a co-stress(drought,salt and alkali)maize mi RNA library constructed in our laboratory,zma-mi R169 c was down-regulated seven-fold,which showed that zma-mi R169 c might be related to the response of maize to abiotic stress.Therefore,we will study the role of zma-mi R169 c and its target gene Zm NF-YA13 from Zheng 58 maize in plant abiotic stress resistance.Results of q PCR indicated that zma-mi R169 c and its target gene Zm NF-YA13 were differentially expressed under drought,salt,alkali,low temperature,abscisic acid(ABA)and K+ deficiency treatments in shoots and roots of maize seedlings.It showed that zma-mi R169 c and its target gene Zm NF-YA13 might play a role in plant abiotic stress resistance.The expressions of zma-mi R169 c and Zm NF-YA13 were observed in root,stem,leaf,tassel,filament and seed of maize,and the higher expression level were observed in tassel,filament and seed.It showed that zma-mi R169 c and its target gene Zm NF-YA13 may be related to the reproductive development of maize.A 319 bp fragment was amplified by PCR,comprising of a 134 bp precursor sequence,102 bp upstream and 83 bp downstream flanking sequences.Blastn showed that the homology of zma-mi R169 c precursor sequence and the pre-mi R169 c of B73 maize from mi RBase was 100%.Maize expression vector ubi:zma-pre-mi R169 c was constructed and successfully transferred to Agrobacterium tumefaciens EHA101.The Zm NF-YA13 gene was amplified by cloning of partial coding region,3'RACE and 5'PCR technology.The full-length c DNA of Zm NF-YA13 is 1147 bp,comprising of a 47 bp 5'UTR,278 bp 3'UTR containing a zma-mi R169 c cleavage site and 822 bp ORF encoding 273 amino acids.The analysis of amino acids showed that it contained a NF-YB/C binding region,a DNA binding region and a intermediate junction region.The amino acid sequence of Zm NF-YA13 had the highest homology with NF-YA from other species was from 39%-72%.Phylogenetic relationship analysis showed that the closest homologue to Zm NF-YA13 was wheat protein Ta NF-YA10.These results showed that the Zm NF-YA13 gene was a member of NF-YA family.Maize expression vector ubi:Zm NF-YA13(ORF),ubi:Zm NF-YA13(ORF+3'UTR)and tobacco expression vector 35s:Zm NF-YA13(ORF)were constructed and transferred into Agrobacterium tumefaciens EHA101 by freeze-thawing method.35s:Zm NF-YA13(ORF)was transferred into tobacco plants via the Agrobacterium mediated leaf-disc method,and 6 transgenic lines were obtained.The transgenic plants were detected by genomic PCR,RT-PCR and semi-quantitative RT-PCR,of which the L3 and L4 lines with higher expression levels were chosen for subsequent physiological experiments.After 1 week of drought stress treatment,transgenic tobacco plants had a more robust root system,higher dry weight,higher fresh weight,higher leaf relative water content and higher Superoxide dismutase enzyme activity as compared to the wild type tobacco plants.Therefore,overexpression of Zm NF-YA13 gene could improve drought tolerance in tobacco.