Cloning and Expression Analysis of an Ammonium Transporter Gene in Pyrus betulaefolia and Its SNP Analysis in Pyrus

An AMT gene named PbAMT1;2 was cloned from seedling of Pyrus betulaefolia using RACE method based on EST sequence.The full length of PbAMT1;2 is 1 811 bp,and contains a complete open reading frame of 1 515 bp,which corresponding DNA sequence has no intron.PbAMT1;2 encodes a 504 amino acid polypeptide,which exhibits 11 transmembrane domains and possesses 1 glycosylation site,3 tyrosine kinase phosphorylation sites and 8 protein kinase C phosphorylation sites.Sequence analysis indicated that PbAMT1;2 had high identity with other plant corresponding genes,in which LjAMT1;2,AtAMT1;2 and LeAMT1;2 were 80.23%,78.68% and 77.80% identical with PbAMT1;2,respectively.Phylogenetic tree analysis showed that PbAMT1;2 belongs to AMT1 sub-family.The results of RT-PCR showed that PbAMT1;2 expressed mainly in roots and very little in stems and leaves.DNA sequences of AMT1;2 ORF region were cloned by high-fidelity Taq enzyme with the DNA templates from six Pyrus plants including P.betulaefolia,P.calleryana,P.bretschneideri,P.pyrifolia,P.ussuriensis and P.communis.The sequencing results showed that DNA sequences length of AMT1;2 ORF region from six Pyrus plants were all 1 515 bp and the nucleotide similarity was 99.48%.According to the calculation result,the polymorphism frequency was 1SNP/34.43bp,the variation degrees of nucleotide and amino acids were 2.90% and 3.57%,respectively.