Gene Cloning of Puroindoline b in Aegilops juvenalis

Puroindoline a(Pina) and puroindoline b(Pinb) are major genes controlling grain hardness in wheat.According to the conserved regions of Pinb gene reported,one pair of specific primers ForB₂ and RevB₁ were designed and used to amplify Pinb from young leaves genomic DNA and developing endosperm RNA of hexaploid Aegilops juvenalis(UUMMDD).Through gene cloning and sequencing,two new alleles of Pinb,Pinb-allele-1 and Pinb-allele-2,were characteri-(zed).Both of them were 360 bp in size,encoding 119 amino acid residues which corresponded to the structure of mature Puroindoline B(Pin B) protein.They contained the tryptophan motif of WPTKWWK and five disulphide bonds formed by ten cysteines,which were specific to Pinb gene in cereal crop.There existed large differences of sequences of Pinb between Ae.juvenalis and Triticum aestivum.Compared with Pinb-D1a from T.aestivum cv.Capitole,the Pinb genes in Ae.juvenalis shared 93.1% and 93.3% homologies in nucleotide sequence and 90.8% and 92.4% homologies in amino acid sequence respectively.There were 11 amino acid mutations in Pinb-allele-1 and 9 in Pinb-allele-2.Southern blot analysis showed that there were two copies of the gene in Ae.juvenalis.Pinb-allele-2 was verified to express in grain endosperm by RT-PCR,but Pinb-(allele-1) not.The results showed that Aegilops contain grain hardness-controlling genes quite different from those of T.aestivum.