Cloning And Expression Regulation Of Membrane-bound NAC Transcription Factors In Zea Mays

Transcription factor plays an important role during the regulation of plant development and stress resistance of functional genes.Among them,specific NAC transcription factor is discovered in recent years in the plant and plays an important role in plant development,morphogenesis,organ separation,leaf senescence,and abiotic stress response.The membrane-bound transcription factor(MTFs)is a special class of transcription factors.Under usual circumstances;it exists in the cell membrane(cell membrane,endoplasmic reticulum and nuclear envelope)structure in the inactive state by membrane-bound form.The membrane-bound transcription factors associate to the cell membranes and remain inactive.When the cells are stimulated,MTFs are released and transported into the nucleus to exercise their functions.Currently,the biological characteristics of MTFs have not been reported in maize.Seven ZmNTL genes are identified and their protein structure characteristics,system evolutionary tree,subcellular localization,transcription activation activity,tissue specific expression and to abiotic stress and hormone response are investigated in this study.Pure lines of transgenic Arabidopsis thaliana over-expressing ZmNTL1,ZmNTL2 and ZmNTL5 were obtained by Agrobacterium-mediated transformation.We studied the effects of ZmNTL1 on the regulation of water flooding stress response and ZmNTL2 on lignin biosynthesis in Arabidopsis thaliana.The main research process and the results of the experiments include:(1)Analysis of sequence and expression of NAC membrane-bound transcription factor in maize7 members of the transmembrane domain were identified from 216 maize NAC transcription factors using bioinformatics methods,and named as ZmNTL1-7.Sequence alignment revealed that all of ZmNTLs had typical NAC domains at N-terminal,and one transmembrane helical domain at C-terminal.Amino acid sequence alignment,the 7 ZmNTLs divided into 4 groups(including 3 genes pairs),respectively,ZmNTL1/5,ZmNTL2/3,ZmNTL6/7 and ZmNTL4.(2)ZmNTLs phylogenyTo illustrate the evolutionary relationship between heterologous NTLs,a phylogenetic analysis was carried out,involving sequences from A.thaliana(18 genes),from rice(five genes),and from foxtail millet(eight genes)and the seven ZmNTLs using MEGA6.0.Five clades were recognized,only one of which was specific to the dicotyledonous species A.thaliana.The seven maize sequences fell into four clades(ZmNTL1/5 into clade III,ZmNTL2/3 into clade I,ZmNTL4 into clade II and ZmNTL6/7 into clade IV).ZmNTL2/ZmNTL3,At NTL4 in the group I,and AtNTL7,ZmNTL4 in group II,ZmNTL1/ZmNTL5,At NTL8 and AtNTL5 in group III,ZmNTL6/ZmNTL7 and AtNTL6 and AtNTL9 in group IV may have similar function in a subgroup of members.Conservative motif analysis by MEME showed the motifs of ZmNTLs were more conserved than in Arabidopsis thaliana.Analysis of evolutionary relationship of ZmNTLs At NACs OsNACs found that ZmNTLs in Arabidopsis with some NAC transcription factor has more recent evolutionary relationships.For example ZmNTL1 and ANAC086,ANAC045,ZmNTL2 and VND6,VND7 and so on have a close relationship.It indicates ZmNTLs have similar function with some NAC transcription factors in Arabidopsis.(3)Sub-cellular localization and transcriptional activation activity of ZmNTLsThe CDS of ZmNTLs genes were cloned from B73 inbred lines,and their GFP transient expression vectors were constructed and transformed into Arabidopsis thaliana protoplasts.The results indicated that six of the seven ZmNTLs were associated with the plasma membrane and ZmNTL6 with the endoplasmic reticulum.Transcriptional activation analysis revealed that all of the ZmNTLs had transcriptional activation.Quantitative detection of ?-galactosidase expression assay showed that ZmNTL2 transcription activation activity was highest,followed by ZmNTL6;ZmNTL4 and ZmNTL7 were relatively weak.(4)The temporal and spatial expression patterns of ZmNTLs:Real-time analysis showed that the expression of ZmNTL1-7 had obvious tissue specificity.ZmNTL1 was expressed in root,stem and leaf,and ZmNTL1 was highest in leaf,and ZmNTL2 was highest in stem.ZmNTL3-7 was mainly expressed in stem.(5)Response pattern of ZmNTLs response to abiotic stresses and hormones:Real-time PCR analysis showed that ZmNTL1-7 response to abiotic stresses and hormone and the expression pattern showed diversity.ZmNTL1-7 were significantly up-regulated in root and stem under ABA and H2O2 stresses,and the expression pattern was similar.Under PEG treatment,the expression of ZmNTL1-7 was significantly reduced in the stem.ZmNTLs were induced to up regulation under MeJA?BR?2,4-D?KT treatments,especially for the most strongly MeJA response;ZmNTL1,-4,-6 had a small amount of induction,ZmNTL2,-3,-5 had no significant change,ZmNTL7 was inhibited under GA treatment.(6)Obtaining transgenic Arabidopsis overexpression lines of ZmNTL1,ZmNTL2 and ZmNTL5The full length and remove trans-membrane domain fragments of ZmNTL1,ZmNTL2 and ZmNTL5 were connected to the over-expression vector pK2GW7 and their transgenic Arabidopsis pure lines seeds were obtained.In normal growth conditions,their root length and seedling had no significant difference between transgenic lines and wild type.(7)Response of 35S::ZmNTL1,-2,-5-?TM to hydrogen peroxide stressThe seeds of 35S::ZmNTL1,-2,-5-? TM and wild type were drawn in 1/2MS solid medium,when the length of root reached to 1cm,they were transferred to containing 0.5mm,1mm,1.5mm and 2mm H2O2 medium on stress treatment.The results show that transgenic lines can obviously relieve the inhibition of hydrogen peroxide,the three genes ZmNTLs response of hydrogen peroxide stress.(8)Role of ZmNTL1 in water flooding stress toleranceUnder the condition of soil moisture saturation,there was no significant difference between ZmNTL1 transgenic Arabidopsis thaliana and wild type.35S::ZmNTL1-?TM growth was weaker than that wild type.ZmNTL1-?TM over-expression lines showed growth retardation,dwarf,leaf curl and other phenotypes under flooding stress.It showed that ZmNTL1 reduced the tolerance of Arabidopsis thaliana to water flooding stress.It showed that the gene expression of AtICL,NADP-ME1,At ADH1 was significantly higher than that of wild type by real-time PCR analysis.ZmNTL1-TM protein can bind to 4 sites of the AtRbohD promoter and showed that the expression of ZmNTL1 directly regulates At RbohD by EMAS(gel retardation assay)analysis.It was found that the growth of rbohd(knockout lines)was inhibited under water,while 35S::AtRbohD was better than the control,which indicated thatAtRbohD was involved in the regulation of water flooding stress in Arabidopsis thaliana.When AtRbohD overexpression vector was transferred into 35S::ZmNTL1-?TM,the sensitive phenotype of 35S::ZmNTL1-?TM is restored under flooding stress.It proved the ZmNTL1 can direct regulation and AtRbohD.In summary,ZmNTL1 as the negative regulatory factor can directly down-regulate the AtRbohD expression to reduce the tolerance to flooding.(9)The role of ZmNTL2 in the regulation of lignin biosynthesis in plantsThe hardness of stem was better than that of wild type,when 35S::ZmNTL2 and35S::ZmNTL1-TM transgenic Arabidopsis growth for 7 weeks.Compared with the wild type,the number of cells in the xylem of the stem xylem which can be stained was increased,and the color was deeper via toluidine blue and phloroglucinol staining.The result indicated that the distribution range and content of lignin was higher in transgenic Arabidopsis than that of the control.The results of in situ hybridization showed that ZmNTL2 was mainly expressed in xylem.Real-time PCR analysis showed that the lignin biosynthesis key marker genes(PAL,C4 H,HCT,CAD,CCR,C3 H,COMT and F5H)in 35S::ZmNTL2 and 35S::ZmNTL1-?TM were significantly up-regulated.These results indicated that ZmNTL2 may regulate lignin synthesis related gene expression to improve the degree of lignification of plant stem.In conclusion,ZmNTL genes play an important role in plant development and response to abiotic stresses,and it is a great significance to excavate the function of ZmNTLs in the analysis of the mechanism of the growth and development in maize.