杜梨PbPEAMT的克隆、序列分析及表达特征

采用RT-PCR、cDNA末端快速扩增法和长片段PCR技术，从杜梨幼苗中获得1个磷酸乙醇胺N-甲基转移酶基因（Pb—PEAMT），运用生物信息学方法分析它的序列特点，并通过跨内含子引物进行半定量RT-PCR研究其在非生物胁迫下的表达情况。结果表明：PbPEA臌因编码区DNA序列长为3320bp，由11个外显子和10个内含子组成，cDNA序列长1479bp，推导的多肽包括2个II型甲基转移酶保守结构域，与蓖麻PEAMT蛋白相似性最高（86％），亲缘关系最近。PbPEA燃因在杜梨幼苗根和叶中均为诱导型表达，100mmol·L^-1氯化钠、10％（w／v）聚乙二醇、180mmol·L^-1甘露醇或20μmol·L^-1脱落酸处理后PbPEAMT表达水平上升，表明PbPEAMT对盐碱、干旱和渗透胁迫存在表达响应，可能参与ABA介导的逆境信号转导途径。

Cloning, Sequence Analysis and Expression Characterization of PbPEAMT Gene in Pyrus betulaefolia Bunge

N-methylation of phosphoethanolamine, the committing step in choline biosynthesis in plants, is catalyzed by phosphoethanolamine N-methyltransferase （PEAMT, EC 2.1.1.103）. Herein we report the cloning and characterization of a novel birch-leaf pear （Pyrus betulaefolia） phosphoethanolamine N-methyltransferase gene （PbPEAMT） using a combination of homologous cloning, PCR and bioinformatics strategy. At the same time, semi-quantitative PCR with cross-intron primers was adopted to study the expression features of this gene under abiotic stresses. The results showed that the DNA sequence of PbPEAMT gene is 3 320 bp, which con- sists of 11 exons and 10 introns. The cDNA sequence ofPbPEAMT gene is 1 479 bp in length and encodes a 492-amino-acid peptide, which includes two conserved motifs of type II methyltransferase. Homology analysis showed that the deduced PbPEAMT protein was highest homologous to castor PEAMT protein （86%）. Furthermore, PbPEAMT was more related to castor PEAMT through phylogenetic analysis. RT-PCR analysis showed that expression of PbPEAMT was induced by 100 mmol.L^-1 NaCl, 10% （W/V） PEG6000, 180 mmol.L^-1 mannitol or 20 p, mol.L^-1ABA treatments. These data indicate that PbPEAMT expression responds to salinity, drought and osmotic stresses, which may be involved in ABA-mediated stress signal transduction pathway.