NAC Gene Cloning, Sequence Analysis And Prokaryotic Expression From High Quality Wheat Variety

NAC transcription factors family, the large number of transcription factor families, is unique to plants. It plays an important role in plant growth and development and stress responses. Studies shown that NAM-B1 gene encoded NAC proteins, is related to wheat grain protein content, zinc, iron content, and the senescence of wheat plant. Therefore, it has an important role in the nutritional quality of wheat. The relevant reports are not many in wheat, and only a preliminary stage.To further study the NAC transcription factors family of wheat, a pair of specific primers is designed, cloned a number of TaNAC gene from a group of high-quality wheat, and successfully expressed the NAC gene of Triticale76 in E.coli. The results as following:1. 34 genes were obtained, and a partial sequence was submitted to GenBank (GenBank Accession No. HQ872050~HQ872051, HQ630371~HQ630375). Sequence analysis shown that, NAC gene has two introns, and the complete open reading frame, ORF size about 1151-1218bp, amino acid sequences were between 383~405. NAC genes encoding amino acid sequence homology is up to 99% with wheat relatives wild emmer wheat, Aegilops tauschii that have been submitted. Therefore, initially identified those are the gene encoding NAC Protein.2. Phylogenetic analysis of all sequences can be roughly divided into 3 types, this inference may be based on wheat chromosome:2B,2D,6B.3. The bioinformatics analysis about the NAC of Shaan 253, shown that: contained a typical domain of NAM and the NAC domain, was alkaline soluble protein, located in the nucleus, without NLS, including several potential modification sites. Nucleotide comparison analysis, NAC gene mutation occurred in intron regions, a few mutation is in the third site of codon, belonging to synonymous mutation. Amino acid sequence analysis revealed Cysteine mutation in the transition region C~D conserved regions, is a rare variation. The disulfide of Cysteine plays an important role in gluten elasticity.4. RNA extracted from triticale 76. By RT-PCR method, obtained a cDNA sequences and successfully constructed the prokaryotic expression vector, named as pET32a-H76-NAC, expressed in E. coli Rosetta gami B (DE3) by IPTG induction. Sequence analysis showed that containing 1212 bp complete coding sequence. Induced with IPTG, the product by Western blot and SDS-PAGE identificatted, there were a 60kD band. Fusion protein proved successful.