Cloning and Expression of Pinb Gene in Aegilops tauschii

Puroindoline a(Pina) and puroindoline b(Pinb) are the major genes controlling grain hardness in wheat.According to the conserved regions of Pinb gene reported,one pair of specific primers was designed and used to amplify one new Pinb allele from genomic DNA of young leaves of Aegilops tauschii(DD).Sequence analysis indicated that it was 447 bp in size,encoding 148 amino acid residues which corresponded to the structure of PinB protein,with the tryptophan motif of WPTKWWK and five disulphide bonds formed by ten cysteines,which were specific to PinB in cereal crop.Compared with Pinb-D1a from Triticum aestivum cv.Capitole,this new Pinb allele in Ae.tauschii contained 14 amino acid mutations,among which there was one mutation adjacent to the tryptophan motif(Val66Phe),sharing 93.3% homologies in nucleotide sequence and 90.5% homologies in amino acid sequence,respectively.The expression of Pinb in grain endosperm was verified by RT-PCR and Western Blot analysis.The results showed that Ae.tauschii contained grain hardness-controlling gene quite different from that of T.aestivum and further study on this gene would benefit our understanding of the mechanism underlying the formation of wheat kernel texture.