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The Roles of NAC Transcription Factors in Responses to Drought Stress in PeanutCN



Abstract:Peanut(Arachis hypogaea)is the main oil crop and cash crop in the worldwide.Drought stress always causes the decline of yield and quality in peanut,and increases the risk of contamination by aflatoxins.With the intensification of global warming and the increasing in demand for peanut,the supply and demand of peanut will become increasingly prominent.Therefore,it is of great significance to reduce the adverse effects of drought on peanut production by improving the drought tolerance of peanut.NAC transcription factor(TF)is one of the most important transcription factors in plants,and it has multiple family members.The majorities of NAC proteins have the highly conserved NAC domain at N-terminal and the highly diversified transcriptional regulatory domain at C-terminal.They are widely involved in plant growth and development,and responding to biotic and abiotic stresses.So far,there is no systematic study on the NAC family in peanut.In this study,the NAC family genes in peanut were systematically studied by using a series of methods,including the phylogenetic analysis,chromosome location analysis,repeated event analysis,transcriptome expression data analysis,and qPCR verification.At the same time,the AhNAC14,AhNAC20 and AhNAC120 genes were selected for further analyses of expression profile and functional characteristics.These results will provide the foundation for the genetic improvement of peanut drought tolerance.The main results are as follows:(1)132 AhNACs were identified from the genome of cultivated peanuts and were divided into 8 subgroups(Ⅰ-Ⅷ)based on their phylogenetic relationship with Arabidopsis NAC proteins and the conserved motifs.These genes are unevenly distributed on all 20 chromosomes,including116 pairs of fragment duplication events and 1 pair of tandem duplication.Transcriptome analysis showed that many AhNAC genes responded to drought and abscisic acid(ABA)stress,especially most members of groups Ⅳ,ⅣI and Ⅷ,which were induced or inhibited in roots or leaves treated with PEG or ABA expression.In addition,the response patterns of 20 AhNAC genes to PEG and ABA treatment were verified by qRT-PCR.Most results of qRT-PCR analysis are consistent with the results of transcriptome analysis.(2)Cloning and sequence analysis of AhNAC14,AhNAC20 and AhNAC120 genes.The ORF of AhNAC14,AhNAC20 and AhNAC120 were cloned.Among them,the AhNAC14 is 751 bp in ORF size,which encodes 211 amino acids,and its theoretical molecular weight(MW)and isoelectric point(p I)are respectively 23.46 kDa and 9.45,and its g DNA contains 2 exons and 1intron;The ORF size of AhNAC20 is 861 bp,the number of encoded amino acids is 286,and its theoretical MW and p I are 32.83 k Da and 8.48,respectively,and similar as AhNAC14,its g DNA also has 2 exons and 1 intron;The ORF of AhNAC120 is 915 bp in size,which encodes 304 amino acids,and its g DNA contains 3 exons and 2 introns,and its theoretical MW and p I are 35.08 kDa and 6.42,respectively.It was predicted that these three proteins are hydrophilic proteins,and there are no possibility of transmembrane structures and signal peptides.(3)Analysis of gene expression patterns of AhNAC14,AhNAC20 and AhNAC120.The expression level of AhNAC14 in different organs showed great differences,with the highest expression level in lateral roots,followed by stems,main roots and flowers,and relatively the lowest expression level in young leaves.Using 20% PEG6000 to treat the two-week-old seedlings,it was found that AhNAC14 showed an overall increasing trend in lateral roots or taproots within24 hours of treatment.Two-week-old peanut seedlings were treated with 75 μM ABA,and it was found that AhNAC14 showed a continuous increase in the lateral roots within the first 8 hours,and rose rapidly between 8-10 hours to reach the peak of expression,and the expression began to decline after 24 hours;while in the taproots,AhNAC14 expression showed a downward trend.The expression levels of AhNAC20 in different organs showed large differences.The expression level of AhNAC20 was the highest in flowers,followed by lateral roots and functional leaves,and the expression level was relatively lowest in stems.After treatment with PEG6000,the expression level of AhNAC20 in the lateral roots continued to increase within 0-24 h,and increased sharply between 24-36 h,and then the expression level began to slowly decrease;in the taproots its expression showed a continuous increase within 48 h under the PEG6000 treatment.Using ABA treatment,the expression of AhNAC20 increased continuously within the first 6 hours in lateral roots,and reached the peak level at 8-24 h,and then began to decline after 24 h;in the taproot,the expression of AhNAC20 showed a downward trend within 48 h.The expression level of AhNAC120 in different organs showed great differences.The expression level of AhNAC120 was the highest in the taproot,followed by lateral roots,stems,functional leaves and young leaves,and its expression level was relatively lowest in flowers.Under PEG6000 treatment,the expression of AhNAC120 in the lateral roots continued to rise within 0-24 h,and increased sharply between 24-36 h,and then the expression began to slowly decrease;the expression of AhNAC120 in the taproot showed an upward trend within 48 h,and the expression reached the highest level at 48 h.Using ABA treatment,it was found that the expression of AhNAC120 increased within the first 10 hours in lateral roots,and reached a peak at 24 hours,and then began to decline;the expression of AhNAC120 in the taproot showed a downward trend within 48 hours.(4)Identification of AhNAC transcription factors.The analysis results of transcription activities by yeast system found that AhNAC14 has no transcriptional activation activity,and AhNAC20 and AhNAC120 have transcriptional activation activity,and their activation domain located at the C-terminus of AhNAC proteins.To explore the subcellular localization of three target genes,three vectors expressing the fused protein of AhNAC and GFP were constructed.AhNAC14 and AhNAC20 located in the nucleus and cytoplasm,and AhNAC120 located in the nucleus.(5)Function analysis of candidate AhNACs genes.Overexpression vectors of the AhNAC14,AhNAC20 and AhNAC120 genes have been constructed.
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    (D) Agriculture

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