Cloning and expression of three lipoxygenase genes from liverwort, Marchantia polymorpha L., in Escherichia coli

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• 作者：Hirosuke Kanamoto ; Miho Takemura ; Kanji Ohyama ; ^{kohyama@ishikawa-pu.ac.jp}
• 关键词：AA ; arachidonic acid ; ALA ; 伪-linolenic acid ; CaMV ; cauliflower mosaic virus ; EPA ; eicosapentaenoic acid ; EST ; expressed sequence tag ; HEPE ; hydroxy eicosapentaenoic acid ; HETE ; hydroxy eicosatetraenoic acid ; HODE ; hydroxyoctadecaenoic acid ; HOTrE ; hydrox
• 刊名：Phytochemistry
• 出版年：2012
• 期刊代码：85_00319422
• 类别：ch
• 出版时间：May, 2012
• 卷：77
• 期：Complete
• 页码：70-78
• 文件大小：1755 K

摘要

Three genes homologous to plant lipoxygenase genes were identified from the EST libraries of Marchantia polymorpha, in order to clarify the function of LOXs in bryophytes. Full-length genes were isolated using 5鈥? and 3鈥?RACE methods and named MpLOX1, MpLOX2, and MpLOX3, respectively. To investigate the enzymatic activities of liverwort LOXs, recombinant MpLOX1, MpLOX2, and MpLOX3 proteins were prepared from Escherichia coli cells expressing the corresponding gene. LC-MS/MS analyses and chiral column chromatography of their reaction products showed that MpLOX1 codes for 11S/15S-lipoxygenase against eicosapentaenoic acid and for 15S-lipoxygenase against arachidonic acid, and that MpLOX2 and MpLOX3 code for 15S-lipoxygenase against eicosapentaenoic and arachidonic acids. Phylogenetic analysis showed that the liverwort lipoxygenase genes separated from the ancestor of higher plants in the early stages of plant evolution. Quantification analyses suggested that arachidonic acid and eicosapentaenoic acid were preferred substrates. Furthermore, each liverwort lipoxygenase exhibited highest activity at pH 7.0 and dependency on Ca²⁺ ion in the oxygenation reaction.