Cloning And Functional Analysis Of ABF Transcription Factor Gene In Nicotiana Tabacum

Drought, low temperature and high salinity are main abiotic stress factors whichinfluence plants’ growth and are direct causes leading to crop losses, to some severeextent, will result in plants’ death. As an important transgenic model organism and maineconomical crops of our country, tobacco hold valuable significance for functionalgenomics research and economic development. Tobacco has a high requirement forwater during the whole growth period. Only under adequate water supply, the quantityand quality of tobacco leaves can be assured. While in agricultural production, tobaccoagriculture of our country suffered huge losses due to the restrictions of geographicalfactors and periodic climate change. Isolation and identification tobacco abiotic-inducedgenes has valuable significance for revealing tobacco adverse-resistance molecularmechanisms and the art of tobacco genetic improvements.ABI5/AREB/ABF transcription factors belong to A subfamily of bZip transcriptionfactors, they specifically exist in plants. They are in a critical position in ABAtransduction pathway and their functions involve signal transduction for drought andhormone. ABI5/AREB/ABF transcription factors recognize and bind to ABRE elementof stress-responsive genes’ promoter, improve plants drought resistance by regulatingstress-related gene expression. therefore, ABI5/AREB/ABF transcription factors acts asa transmission switch in abiotic signal transduction process, playing an important role inplant adversity resistance. To reveal tobacco molecular mechanism of abiotic resistanceand cultivate high-quality tobacco varieties, we had successfully isolated two tobaccoABF genes, NtABF1and NtABF2, and made a comprehensive evaluation including theirexpression profiles, the recongnition specificity of promoters and the capacibility ofabiotic resistance of transgenic plants, to provide more references for revealing theirrole and action mechanism in plant adversity resistance. The main research results areas follows:①Cloning and bioinformatics analysis of tobacco NtABF1and NtABF2geneWe successfully cloned two ABF genes, NtABF1(GenBank accession numberKF736849) and NtABF2(GenBank accession number KF736850), from tobacco cultivar“honghuadajinyuan”. Bioinformatics analysis results indicate that, the full length ofNtABF1Open reading frame(ORF) is999bp, encoding332amino acids whosemolecular weight is36.45kDa and theoretical isoelectric point pI is9.04; The full length of NtABF2ORF is1305bp, encoding433amino acids whose molecular weight is46.92kDa and theoretical isoelectric point pI is9.35. Both NtABF1and NtABF2arehydrophilic proteins holding BRLZ conservative domain and neither of them has signalpeptide nor transmembrane structure domain. Alpha helix and random coil are mainadvanced structures of NtABF1and NtABF2, beta turn and extended strand are less. Onthe subcellular level, both NtABF1and NtABF2localized to nucleus. Phosphorylationsite analysis indicates, they both have Ser, Thr, Tyr kinase recongnition sites.Phylogenetic analysis and multiple sequence alignment results indicate that, NtABF1and AtAREB3have closer evolutionary relationship and share sequence similarity to54.49%; NtABF2has closer genetic distance to SlAREB1, StABF1and StABF, andtheir sequence similarity are83.63%,83.52%and83%, respectively. Functionalprediction results indicates, both NtABF1and NtABF2are transcription factors.②Expression profiles analysis of tobacco NtABF1and NtABF2Analysising the tissue expression profile of NtABF1and NtABF2indicates, bothNtABF1and NtABF2sharing the same expression pattern, both of them has the highesttranscripts in roots, sequently is stems, leaves, flowers. And in each tissue, NtABF1hashigher relative expression level than NtABF2. Expression profile analysis of NtABF1and NtABF2under adverse condition indicate that, NtABF2is easier induced bydifferent adverse conditions: NtABF2expression level was up-regulated by PEG,dehydration, NaCl, low temperature and ABA while was down-regulated by MeJA;NtABF1expression level was up-regulated by PEG, NaCl, low temperature and ABA,while was down-regulated by dehydration and MeJA.③Binding specificity analysis of NtABF1and NtABF2protein with ABREelement in vitroWe successfully constructed prokaryotic expression vectors for NtABF1andNtABF2, pET28a-NtABF1and pET28a-NtABF2, transformed into expression strainsTransRosetta(DE3), target proteins were induced and purified, respectively. EMSAassay was used to detect binding capacibility between target protein and C-box, G-boxABRE element in vitro. Electrophoresis results indicate that both NtABF1and NtABF2protein can bind to C-box ABRE element specifically. The binding ability of NtABF2with C-box ABRE element is strong, the binding signal of NtABF2with G-box elementcan be detected when NtABF2protein concentration is improved.④Obtaining of overexpression transgenic plants and functional identification oftobacco NtABF1and NtABF2 We successfully constructed plants’ overexpression vector for tobacco NtABF1andNtABF2, pCXSN:35S:NtABF1and pCXSN:35S:NtABF2, transformed intoAgrobacterium tumefaciens LBA4404and then transformed into plant leaf tissues bymeans of leaf discs method, and isolated19positive transgenic lines for NtABF1andNtABF2, respectively through PCR. After30days with water control, relative transcriptlevel of target gene(NtABF1or NtABF2) and stress-induced genes for transgenic lines7#,8#,10#of pCXSN:35S:NtABF1and transgenic lines11#,14#,17#ofpCXSN:35S:NtABF2were analysised. The results indicates that NtABF1relativetranscript abundance of7#,8#,10#for pCXSN:35S:NtABF1was significantly higherthan WT, and NtAPX and NtERD10C expression level increased in7#and8#. While theexpression level of NtNCED1was suppressed in the three transgenic lines. Infering thatthe drought resistance of the three lines may be improved though other ways such asscavenging ROS and inducting the expression of adverse-resistance proteins.17#transgenic lines of pCXSN:35S:NtABF2has a higher transcript abundance for NtABF2,NtAPX and NtNCED1. While relative expression level of NtERD10C was lower thanWT in11#,14#and17#lines. It can be inferred that T0generation of17#may has abetter comprehensive drought resistance than14#and17#.