Cloning And Function Analysis Of ZmNAOD In Maize

Maize in one of the most important crops for food and feeds.Seed development and photoperiod sensitive are the key points that will influence the yield.N2-acetylornithine deacetylase(NAOD)is an important enzyme in L-arginine biosynthesis pathway,which catalyzes the reaction of N2-acetyl-L-ornithine and H2 O,with acetate and L-ornithine as products,and is involved in many processes including fruit development,response to abiotic stresses and photoperiodic regulation.In this research,three previous materials,Zheng58(receptor parent),Chang7-2(donor parent)and Z22(single segment substitution line),were used.In the previous study,a major QTL of hundred kernel weight,qhkw5-3,was mapped between makers SYM033 and SYM108.In this interval,a NAOD coding gene was found as GRMZM2G181273,which might be related to kernel development.GRMZM2G181273,named Zm NAOD,was taken as the target gene in this research.Expression analysis,promoter analysis,subcellular localization and transgenic methods were applied to identify the function of Zm NAOD.The main results are as follows:1.A c DNA sequence fragment of Zm NAOD was obtained via PCR from inbred Chang7-2,Zheng58 as well as from the inbred Z22,all of which contained a CDS of 1344 bp encoding for 447 amino acids.And there was no difference among them.Bioinformatics analysis showed that NAOD in mazie,C2195H3441N585O661S18,is a hydrophilic protein which has a molecular wright of 49.179 k D,a theoretical isoelectric point of 5,45 and 18 phosphorylation sites,but it has no signal peptide sequence or transmembrane domain.It has 36.42% of alpha helix,34.90% of random coil,19.91% of extended strand and 8.95% of beta turn in the secondary structure.Amino acide sequence analysis indicated that Zm NAOD has a conserved Peptidase_M20 domain.Phylogenetic analysis indecated that Zm NAOD coding protein has similarity of 97% to sorghum NAOD protein,and the similarity of At NAOD(At4g17830)protein was 60.54%.2.All the DNA sequences contain 10 exons and 9 introns.And Chang7-2 and Z22 showed no difference in the DNA sequences,while Zheng58 has a 40-bp-deletion and 4 SNP in the 3’UTR after stop codon.3.The tissues expression analysis showed that in Zheng58,the tassel has the most amount of Zm NAOD accumulation,and then is the stem,root,leaf,kernel in order of expression level,respectively.The order of expression level from the most to the least in Chang7-2 is tassel,kernel,stem,leaf and root;while the order in Z22 is kernel,tassel,root,stem and leaf.And during the different phases of kernel development,Chang7-2 and Z22 showed the same tendency: they increased rapidly from 0 DAP to 15 DAP,and then decreased significantly.While in Zheng58,the expression of Zm NAOD increased continually from 0 DAP to 20 DAP,and reached a peak at 20 DAP then decreased significantly.These results demonstrated that Zm NAOD might be associated with the regulation of kernel development in maize.4.Three 2401 bp promoter regions were gotten from Zheng58,Chang7-2 and Z22.The sequence alignments showed that Zheng58 has 2 SNP with Chang7-2 and Z22,while the last two were the same.Plant CARE was used to predict the cis-element in the promoter region.Cis-regulatory elements involved in endosperm expression(GCN4-motif and Skn-1_motif),element involved in circadian control(Circadian),light responsive elements(G-box、GA motif、GAG motif、I-box)exist in the promoter region,and some hormone responsive elements and stress responsive elements were also found.Chang7-2 has 1 ARE element and 1 HSE elements more than Zheng58.5.The subcellular localization of Zm NAOD protein in the root of Arabidopsis seeding showed the green fluorescent protein gathered in the cytoplasm and plasma membrane,which suggested the NAOD protein in maize is located in the cytoplasm.And the result is the same as the subcellular localization prediction.6.The overexpression vector p CAMBIA1304-Zm NAOD promoted by Ca MV 35 S was constructed and transformed into Arabidopsis.The expression of Zm NAOD was higher in root and seqlice in homozygous progenies of transgenic lines.Further study showed that the length of root of ectopic expression seedlings increased significantly than that of the WT plants after 10 days in the dark cultivation.Three transgenic lines T3-17,T3-23 and T3-26 which had more Zm NAOD accumulation than other lines were used for phenotype identification.The date of first buds visible and the date of first flower were significantly earlier than WT.And compared with the wild type plants,the 1000-seed weight of the transgenic lines increased significantly as well as the seed length.The results suggested that Zm NAOD might increase seed size and wight,and might influence the photoperiod regulation in Arabidopsis,and light might have influence on the regulation of Zm NAOD in plant growth and development.