杜梨铵转运蛋白基因的克隆表达及在梨属植物中的SNP分析

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

利用EST并结合RACE方法从杜梨幼苗克隆获得1个AMT基因(PbAMT1;2).分析显示,PbAMT1;2cDNA全长1 811 bp,开放阅读框为1 515 bp,其对应基因组DNA序列不含内含子.PbAMT1;2编码的蛋白由504个氨基酸组成,具有11个跨膜域,1个N-糖基化位点、3个酪蛋白激酶磷酸化位点和8个蛋白激酶C磷酸化位点.同源性分析发现,PbAMT1;2与其他植物的AMT具有较高的一致性,其中与百脉根LjAMT1;2的一致性为80.23%,与拟南芥AtAMT1;2的一致性为78.68%,与番茄LeAMT1;2的一致性为77.80%.系统进化树分析表明,PbAMT1;2属于AMT1亚家族.半定量RT-PCR结果显示,PbAMT1;2主要在根部表达,而在茎和叶中几乎没有表达.以杜梨、豆梨、砂梨、白梨、秋子梨和西洋梨等6种梨属植物的DNA为模板,高保真Taq酶PCR扩增AMT1;2基因ORF区DNA序列,发现6种梨属植物的AMT1;2 ORF区DNA序列长度均为1 515 bp,相似性高达99.48%,但在44个核苷酸位点中存在SNPs,导致18个氨基酸位点发生变异,多态性频率为1SNP/34.43 bp,核苷酸变异度为2.9%,氨基酸变异度为3.57%.

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.