Isolation And Functional Analysis Of Transcriptional Factor NtbZIP66 Under Drought Stress

·bZIP?basic region/leucine-zipper motif?is one of the largest transcription factor families in plants and is widely involved in all kinds of plant stress.In laboratory transcriptome analysis of tobacco K326 under drought stress,it was found that NtbZIP66gene was significantly down-regulated under drought stress.In this study,the NtbZIP66gene of tobacco bZIP family was cloned with K326 as the material.The expression patterns of this gene in tobacco under drought,low temperature,NaCl and NaHCO3 stress were detected by q-PCR.To analyze the function of NtbZIP66,the overexpression vector of pCAMBIA1301-NtbZIP66 was constructed.Using the method of Agrobacterium tumefaciens-mediated transformation of the Nicotiana benthamiana.The overexpressed plants were treated with drought stress,phenotypic and physiological and biochemical indices of wild-type and overexpressed plants were observed.The main research results are as follows:1.The CDS sequence of NtbZIP66 gene was cloned,with a total length of 915bp and coding 304 bases.The physical and chemical properties of NtbZIP66 protein were analyzed by online software ProtParam.The results showed that the molecular formula of NtbZIP66protein was C₁₄₄₇H₂₂₈₇N₄₃₁O₄₇₀S₁₀,the molecular weight was 33.562kDa,the theoretical isoelectric point was 5.92,the fat index was 65.86,and the stability coefficient was67.47.Through further analysis of amino acids encoded by NtbZIP66 gene,it was found that NtbZIP66 protein contained a total of 20 amino acids,of which Ser content was the highest,accounting for 10.90%of the total amino acids,followed by Arg,Pro and Asn,Trp and Tyr content was the lowest,only accounting for 0.70%of the total amino acids.Thirty-one amino acid residues?Arg+Lys?were positively charged and 36 amino acid residues?Asp+Glu?were negatively charged in NtbZIP66.The evolutionary tree showed that NtbZIP66 had a high homology with CabZIP1 and ZmbZIP60,which were classified as a cluster.Cis-acting elements in the promoter region indicate that the promoter sequence of NtbZIP66 contains abscisic acid response elements,suggesting that NtbZIP66 may be associated with stress.2.Tissue specific expression of NtbZIP66.NtbZIP66 was expressed in the root,stem,leaf in tobacco and the highest expression was found in roots.3.Expression patterns of NtbZIP66 under different abiotic stresses.The expression level of NtbZIP66 was significantly down-regulated under 15%PEG6000 and low temperature?4??treatment,and the expression level was basically unchanged under the stress of high salt?200mmol/L NaCl?and alkali?100mmol/L NaHCO₃?,suggesting that NtbZIP66 was related to drought and low temperature stress of tobacco.4.The overexpression of NtbZIP66 reduced the drought tolerance of tobacco.Based on pCAMBIA1301,an overexpression vector pCAMBIA1301-NtbZIP66 containing the target fragment of NtbZIP66 gene was constructed.The genetic transformation of tobacco was mediated by agrobacterium tumefaciens LBA4404 to obtain overexpressed plants.Of wild type plants with different stress treatment and gene expression,natural dry after treatment with drought stress PEG6000,overexpression of wilting degree is higher than the wild type plants,leaf malondialdehyde content is higher,lower leaf proline content,less leaf antioxidant enzyme activity,leaf soluble protein content is lower and suggest NtbZIP66gene plays an important role in the tobacco drought relief way.