| Aegilops tauschii and Secale cereale,with some excellent characteristics such as disease resistance,cold resistance and high protein content,were always used as exogenous gene donor in wheat genetic improvement.Prolamin-box binding factor(PBF),a DOF transcription factor,was involved in seed storage protein expression and thus influenced the processing quality of wheat flour.So far,the detailed sequence information about Pbf genes had been just restricted in common wheat Chinese Spring,but there were no relevant reports in Ae.tauschii and S.cereale.To provide candidate genes for the wheat quality improvement,two pairs of gene specific primers were designed and applied to isolate genes from two Ae.tauschii(KJ544771,KJ544772)and twelve S.cereale(KJ747373-KJ747384),respectively.After sequence analysis,two different Pbf genes(KJ747373,KJ747378)from S.cereale were picked out for prokaryotic expression,affinity purification and further quality effect appraisal.The results were as follows:(?)Fourteen pieces of Pbf genes were cloned including two from Ae.tauschii(AS90 and AS2386)and twelve from S.cereale(rye01-ryel2).The genes from Ae.tauschii were 993 bp long and encoded 330 amino acids.But the ones from S.cereale were 987 bp long and encoded 328 amino acids.(?)Nucleotide sequence analysis showed that the Pbf genes from Ae.tauschii and S.cereale were relatively conserved,respectively.Four from S.cereale including KJ747377,KJ747378,KJ747380 and KJ747382 were identical.And further alignment with Pbf genes from common wheat and its ancestors indicated that Pbf genes from Ae.tauschii with those from common wheat and its ancestors were more conserved.KJ544772 from Ae.tauschii was identical to KC849701 from common wheat Chinese Spring.But there existed more extensive variations for Pbf genes from S.cereale than those from others.In addition to the point mutations distributed in the entire sequence,there was also a continuous delete mutation of six bases in the regulation region of PBF.(?)Sequence analysis of amino acids showed that PBFs from Ae.tauschii and S.cereale belonged to alkalescent and hydrophilic proteins and localized themselves in nucleus.And all of them contained typical domains of DOF.Further alignment with PBFs from other species including barley,common wheat and its ancestors showed that non-redundant amino acids in DOF domain(36aa-90aa)were extremely conserved.The conserved sequences of nuclear localization signal(NLS,26aa-29aa)and serine-rich stretches(Ser Hinge)located downstream of the DOF domain from barley and S.cereale were identical,and those from common wheat and its ancestors were relatively conserved.But there existed stable single amino acid mutation between above two groups.The asparagine-rich region in the C-terminal domain for transcriptional regulation existed significant mutations among different species.PBFs from common wheat and its ancestors near this region lack three amino acids continuously compared to that from barley.However,PBFs from S.cereale lack two amino acids continuously compared to those from common wheat and its ancestors near this region.(?)The phylogenetic analysis indicated that the PBFs were species-specific.Among these PBFs,the ones from common wheat had a closer relationship with the one from AS2386 than that from AS90,while the PBFs from Ae.tauschii and common wheat were closer in relationship than those from Triticum urartu and common wheat.Besides,the relationship between PBFs from S.cereale and Hordeum vulgare was closer than that among PBFs from S.cereale,common wheat and its ancestors.(?)Protein analysis by SDS-PAGE indicated that Pbf genes(KJ747373,KJ747378)from S.cereale were expressed successfully in prokaryotic system.Moreover,the isolated PBF by affinity purification with Ni-NTA Resin was added to the basic flour and mixed.And the results showed that both PBFs had a positive effect on wheat processing quality,while KJ747373 was better. |
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