Cloning And Functional Analysis Of A DREB1/CBF Transcription Factor From Zea Mays L.

maize (Zea mays L.) is an important food and economic crop and currentlyproduced in most countries in the world. This plant is used to produce grain andfodder that are the basis of many foods, feed and industrial products. However, theadverse environmental factors, such as drought, cold and soil salinization, seriouslyaffect the growth and development of maize which bring terrible loss to it. Thedehydration responsive element binding proteins (DREB) are important transcriptionfactors that induce a set of abiotic stress-related genes and impart stress endurance toplants. These proteins specifically bind to the DRE/CRT sequence and activate thetranscription of genes driven by the DRE/CRT sequence in plant. In this study, wecloned and characterized a novel gene encoding a DREB1/CBF transcription factorfrom maize B73, named ZmDREB1B. The main results of this study are as follows:1. I designed specific primers based on the sequence from plant transcriptionfactor datebase(PlnTFDB)and obtain the full-length sequence of the ZmDREB1Btranscription factor gene by PCR in maize (B73) genomic DNA. The DNA sequencesof ZmDREB1B comprised a783bp ORF, encoding a putative protein of260aminoacids with a predicted molecular mass of28kDa and isoelectric point (pI) of5.11, nointron. The deduced a mino acid sequences of ZmDREB1B contain a highly conservedAP2/ERF domain, and the result of analysis of software online suggested the proteinwas targeted to the nucleus. Phylogenic analysis further confirmed that ZmDREB1Bbelongs to the A-4subgroup of the DREB/CBF subfamily.2. Expressional analysis of ZmDREB1B in E.coli B21by SDS-PAGE suggestedthe recombinant protein was induced after IPTG treatment and showed higher molecular mass than estimated molecular mass because of the presence of His-tag.The recombinant protein reached maximum at8h～12h. It is the basis of furtherresearch.3. ZmDREB1B were expressed in yeast AH109to analyze their transcriptionalactivation activity. The results suggested ZmDREB1B had transcriptional activationability in yeast which indicated it is a transcription activator.4. The ZmDREB1B∷eGFP fusion gene was obtained by overlapping PCR andcloned into plant expression vector and transformed into onion epidermal cells usingAgrobacterium-mediated transient expression. The results suggested that theZmDREB1B protein was targeted to the nucleus.5. The expression patterns of ZmDREB1B was analyzed using qRT-PCR. Theresults indicated ZmDREB1B had no tissue-specific expression characteristics. Theexpressions was higher in roots than in other tissues. ZmDREB1B transcriptsaccumulated greatly in leaves in response to drought, salt, cold, and ABA, butapplication of exogenous SA and MeJA had no effect on the transcription ofZmDREB1B.6. To investigate the stress tolerance of ZmDREB1B proteins, I constructedprokaryotic expression vector and yeast expression vector respectively and thentransformed them into E.coli BL21and yeast INVScl. The result suggestedZmDREB1B proteins enhanced stress tolerance of E.coli cells and yeast cells.7. ZmDREB1B gene was cloned into plant expression vector pCHF-1301underthe control of the CaMV35S promoter. The pCHF-1301-ZmDREB1B recombinantplasmid was introduced into Arabidopsis by using a vacuum infiltration method. T3generation transgenic Arabidopsis was used to assess stress tolerance of transgenicplants. Quantitative real-time PCR method was employed to analyzed the expressionlevel of some target genes of the DREB1/CBF transcription factor. The resultsindicated overexpression of ZmDREB1B increases tolerance to cold, drought, and high salt in transgenic Arabidopsis. The expression level of some target genes of theDREB1/CBF transcription factor was significantly upregulated in in35S:ZmDREB1Btransgenic Arabidopsis compared with that in the wild-type Arabidopsis.