Cloning,Expression And Preliminary Function Study Of The NAC Transcription Factors ZmNAC19 And ZmNAC31 In Maize

NAC is a specific transcription factor to higher plants,which plays an important role in plant growth and development?anther dehiscence and stress.During the growth period,anther could not crack due to high temperature,high humidity and other environmental factors,which seriously affected maize yield.NAC genes in Arabidopsis thaliana not only participate in leaf development but also regulate anther dehiscence.However,there were few studies on NAC genes in maize.To elucidate the function of NAC genes in regulating leaf and anther development in maize,we screen NAC genes in maize,and Zm NAC19 gene was selected to be mainly expressed in leaves,while Zm NAC31 gene was mainly expressed in tassels and leaves.We performed bioinformatic analysis of Zm NAC19 and Zm NAC31,then constructed the GFP fusion expression vector,overexpress vector and transgenetic Arabidopsis line in which Zm NAC19 and Zm NAC31 were overexpressed.The main results are as follows:1.By comparing the NST protein sequences of Arabidopsis thaliana,100 NAC genes were screened out from maize,and 33 genes were screened out according to the transcriptional data.Finally,Zm NAC19 gene express in leaves and Zm NAC31 gene express in tassels by RT-PCR.Through bioinformatics analysis of the promoter sequences of Zm NAC19 and Zm NAC31,the promoter regions of these two genes have elements of NAC transcription,especially the regulatory elements associated with secondary wall thickening,indicating that these two genes may be involved in the secondary walls thicken of plants.The prediction of the signal peptide and transmembrane structure of Zm NAC19 and Zm NAC31 revealed that these two genes have no signal peptide and transmembrane structure,suggesting that these two genes might be nuclear proteins that play a role in the nucleus.2.We analyzed the expression patterns of Zm NAC19 and Zm NAC31 genes for B73 materials,and the results of RT-PCR and Real-time q RT-PCR were consistent.Zm NAC19 gene was specifically expressed in leaves,and Zm NAC31 gene was specifically expressed in stage11 of anther before earing.3.In this experiment,the subcellular localization of Zm NAC19 and Zm NAC31 protein were studied by transient expression method.By constructing the Zm NAC19::GFP and Zm NAC31::GFP fusion expression protein and using confocal microscopy,the results showed that Zm NAC19 and Zm NAC31 protein were located in the nucleust.4.In order to further clarify the biological functions of Zm NAC19 and Zm NAC31 genes,we constructed the overexpression vectors of Zm NAC19 and Zm NAC31.We use the Agrobacterium-mediated method to obtain transgenic Arabidopsis.Through herbicide screening and DNA molecular level detection,the transgenic T1 generation Arabidopsis thaliana was obtained.Through observation of the overexpressed transgenic T2 generation Arabidopsis thaliana plants,it was found that the Arabidopsis thaliana plants overexpressed Zm NAC19 gene were stronger than the wild type,larger leaves than WT and leaves were curly.This is similar to the Arabidopsis phenotype of overexpression of NST1 and NST2 genes.The overexpression of Zm NAC31 gene in Arabidopsis thaliana was not significantly different from the wild type,because the expression pattern of Zm NAC31 gene showed that the gene was mainly expressed in anthers,followed by leaves.Therefore,the phenotype of Zm NAC31 overexpressed plants in seedling stage is not obviously,to some extent is reasonable.And the role of Zm NAC31 overexpressed Arabidopsis thaliana in anther dehiscence needs to be further observed.Therefore,it is speculated that Zm NAC19 gene may be involved in the regulation of leaf development,and Zm NAC31 gene may be mainly involved in the regulation of anther development.And it is possible that the regulation in the leaves plays an auxiliary role in the regulation in the anthers.