Genetic Analysis Of Wheat TaTGA1 And TaNAC29 Transfaction Factor Gene Cloning And Function Dissection

Transcription factor（TF）is a trans-acting factor,as an important tool,will play a vital role in the research of post-genomics era.The basic region/leucine zipper（bZIP）transcription factors and NAC TFs are pivotal regulators involved in plant growth,biotic stress and abiotic stress.They are important transcription factors in Arabidopsis and rice etc.Many bZIP and NAC genes have been reported with roles in response to biotic stresses and abiotic stresses,Nevertheless,few reports on the bZIP and NAC genes related biotic stresses and abiotic stresses in wheat.To further analysis and dissection the bZIP and NAC TFs function,in this study,we conducted research concerning the function of wheat bZIP and NAC transcription factor involved in biotic and abiotic stress tolerances,in which they provide candidate genes to clarify their mechanism in the process of wheat cultivar,and thus to enrich gene resource library of wheat resistance,and to provide basic data and references sequences for molecular mechanism of biological resistance of wheat.Additionally,these bZIP and NAC genes can be used for marker-assistant selection to develop wheat molecular breeding in the future.Based on data normalized by RMA from wheat genechip and plant transcription factor database,we selected TGA（bZIP type）and NAC probe set.Using in silico cloning technology rapidly Amplification and separate the bZIP and NAC gene,and used bioinformatics to predict their structure and function.Using quantitative real-time PCR to identification of expression characteristics and patterns of the bZIP and NAC TFs,then to further reveal their action mechanism in the process of resistance.The main results are included as follows:1.Using plant transcription factor database and wheat genechip database,we choice 66EST probe sets of TGA and 41 EST probe sets of NAC in wheat genome,homology-based cloning was be used to TGA（Ta.5410.2.S1_aat and Ta.1612.1.S1_at）and NAC（TaAffx.122104.1.S1_sat）probe set separately in N9134 and Zhengmai 9023,respectively.We get 2 TFs,TaTGA1 was confers a high level of resistance against of Bgt races,and TaNAC29 was confers a high level of resistance against of salt and drought.2.We previously identified a pathogen-induced specific-regulated gene TaTGA1 of wheat line N9134 that was induced by powdery mildew（Blumeria graminis f.sp.tritici;Bgt）race E09.To further understand its role in wheat defence,here,we identified it as a bZIP tanscription factor and characterised 2 wheat TGA paralogous,and named as TaTGA1a and TaTGA1b.TaTGA1a consisted of 2547 nucleotides,while the length of TaTGA1b is 1818 bp.Intriguingly,both of them encode a polypeptide of 312 amino acids.3.The rigorous alignment results showed that TaTGA1a and TaTGA1b were different transcribed from 4AS-5969591 and 4BL-7004698,respectively.The genome DNA of TaTGA1a consist of eight intron and nine exon,while TaTGA1b contains nine intron and ten exon.Transcription analys is indicated that TaTGA1a and TaTGA1b were involved in plant defence against Bgt infection.However,TaTGAs was opposite regulated in immune hexaploid wheat at early stages of post-inoculation with Bgt E09.The BlastX alignment result of TaTGA1 showed that it similar with TGA-type bZIP from Aegilops tauschii（XP020199874.1）although a peptide including 22 aa was truncated in N terminal,which demonstrated that it was a novel transcription factor gene in wheat.The estimated theoretical molecular weight of the polypeptide was 34.84 k Da and the pI was 8.94.The basic-leucine zipper domain was located at positions 24-87aa,and the transcription factor TGA like domain was at 110-185 aa.4.To identify which NAC genes may be involved in salt stress response,we analyzed the Affymetrix expression data from studies on wheat gene expression responses to salt stress（E-MEXP-971）,and found that one NAC gene corresponding to the Affymetrix probe set TaAffx.122104.1.S1_sat was up-regulated in salt-stressed wheat plants.The gene was Amplified and sequenced.Sequence alignment revealed that it shows highly homologous to Aegilops tauschii NAC29,and consequently designated as TaNAC29（KP657687）.The gene encodes 298 amino acids and shares high sequence homology with Triticum urartu NAC29（TuNAC29）and Triticum aestivum NAC69（TaNAC69）at the amino acid levels（81%and 85%identities,respectively）.5.To further investigate whether TaNAC29 gene is regulated by salt stress and other treatments,RT-PCR was employed to detect the gene expression profiles.As expected,TaNAC29 gene was obviously induced by salt.The TaNAC29 gene transcripts gradually increased and reached its highest level at 12 h,and then decreased under NaCl treatment.Likewise,TaNAC29 gene was induced to express at the highest levels at 12 h after ABA treatment.Moreover,the gene was also up-regulated by PEG treatment,and reached to its peak value at 24 h after treatment.These results revealed that TaNAC29 transcripts were strongly induced by salt,PEG and ABA.6.To analyze the transcription activation ability of TaNAC29,an autonomous activation test was performed in yeast system.TaNAC29 was fused to the binding domain（BD）of yeast transcription factor GAL4,and transferred into yeast strain AH109.On minimal synthetic dextrose（SD）medium lacking Trp,yeast strains with both BD and BD-TaNAC29grew well.However,on double nutrition-deficient SD medium(SD/-Trp-His or SD/-Trp-Ade,only AH109 strain with BD-TaNAC29 could grow well.The results indicated that TaNAC29could activate the HIS3 and ADE2 reporter genes in yeast strain AH109.The results suggested that TaNAC29 has a trans-activation activity.Enhanced salt tolerance in TaNAC29 transgenic lines compared to WT prompted us to detect the physiological differences.Therefore,the activities of 2 significant antioxidant enzymes were measured in the leaves from potted plants.In normal growth condition,there were no significant differences in H₂O₂,MDA,SOD,POD,APX and CAT activities.However,after 7 days salt treatment,the two transgenic lines displayed significantly higher SOD,POD,APX and CAT activities than WT.These results suggested that over-expression of TaNAC29 reduced ROS accumulation by enhancing SOD,POD,APX and CAT activities under salt stress.