杜梨PbPEAMT的克隆、序列分析及表达特征

李慧1,2, 丛郁3, 常有宏1,2,*, 蔺经1, 盛宝龙1
1江苏省农业科学院园艺研究所, 南京210014; 2国家农业科技华东(江苏)创新中心——高效园艺作物遗传改良实验室, 南京210014; 3中国科学院南京土壤研究所土壤与农业可持续发展国家重点实验室, 南京210008

通信作者:常有宏;E-mail: cyh@jaas.ac.cn;Tel: 025-84390224

摘 要:

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: PbPEAMT基因编码区DNA序列长为3 320 bp, 由11个外显子和10个内含子组成, cDNA序列长1 479 bp, 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。PbPEAMT基因在杜 梨幼苗根和叶中均为诱导型表达, 100 mmol•L-1氯化钠、10% (W/V)聚乙二醇、180 mmol•L-1甘露醇或20 µmol•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。

关键词:杜梨; 磷酸乙醇胺N-甲基转移酶; 基因克隆; 表达特征

收稿:2011-12-14   修定:2012-02-29

资助:国家自然科学基金(31101529)和江苏省农业科技自主创新 资金[CX(11)4050]。

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

LI Hui1,2, CONG Yu3, CHANG You-Hong1,2,*, LIN Jing1, SHENG Bao-Long1
1Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2Efficient Horticulture Crop Genetic Improvement Laboratory, National Agricultural Science and Technology Jiangsu Innovative Center, Nanjing 210014, China; 3State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China

Corresponding author: CHANG You-Hong; E-mail: cyh@jaas.ac.cn; Tel: 025-84390224

Abstract:

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 consists of 11 exons and 10 introns. The cDNA sequence of PbPEAMT 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 µmol·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.

Key words: birch-leaf pear (Pyrus betulaefolia); phosphoethanolamine N-methyltransferase; gene cloning; expression feature

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