《植物生理学报》 2016, 52(5): 659-668

桃CYP707A家族基因的克隆以及表达分析

王东岭, 杜培勇, 郇蕾, 肖伟, 陈修德, 李玲^*, 高东升^*

山东农业大学园艺科学与工程学院, 作物生物学国家重点实验室, 山东省果蔬优质高效生产协同创新中心, 山东泰安271018

通信作者：李；E-mail: lilingsdau@163.com, dsgao@sdau.edu.cn

摘　要：

从油桃‘中油四号’中克隆了3个CYP707A家族成员(PpCYP707A1、PpCYP707A2和PpCYP707A3)的全长, 测序发现其CDS序列与桃基因组公布的序列一致。同源比对发现CYP707A家族在不同物种间具有高度的序列保守性, 并且都含有CYP707A家族基因比较保守的血红素铁配位体半胱氨酸残基; 系统进化树分析表明, PpCYP707A1与马铃薯CYP707A1、花生CYP707A1、菜豆CYP707A1、菜豆CYP707A2的亲缘关系较近, 桃CYP707A2与拟南芥CYP707A2的亲缘关系较近, 桃CYP707A3与拟南芥CYP707A4、花生CYP707A2的亲缘关系较近。荧光定量PCR分析表明, PpCYP707A1在茎中的表达量较高, PpCYP707A2在叶中的表达量较高, PpCYP707A3在花中的表达量较高, 并且桃CYP707A家族一般在成熟组织中的表达水平要高于幼嫩组织。推测桃CYP707A家族成员交叉调控花芽休眠进程, 并且发现花芽中的PpCYP707A2可以被ABA和GA诱导。原核诱导并纯化PpCYP707A1和PpCYP707A2蛋白可以为后续蛋白的功能鉴定奠定基础。

关键词：桃; ABA8’羟化酶; CYP707A; 基因克隆; 表达分析; 花芽休眠; 原核表达

收稿：2015-12-23   修定：2016-04-27

资助：国家自然科学基金项目(31372050)和山东省现代农业产业技术体系水果产业创新团队-栽培与设施装备(SDAIT-03-022-05)。

Molecular cloning and expression analysis of CYP707A gene family in peach

WANG Dong-Ling, DU Pei-Yong, HUAN Lei, XIAO Wei, CHEN Xiu-De, LI Ling^*, GAO Dong-Sheng^*

Shandong Collaborative Innovation Center for Fruit and Vegetable Production with High Quality and Efficiency, State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, China

Corresponding author: LI Ling; E-mail: lilingsdau@163.com, dsgao@sdau.edu.cn

Abstract:

Three members of CYP707A gene family were cloned from ‘Zhongyousihao’ peach. The obtained CDS was consistent with the ones released with peach genome. Multiple sequences alignment showed that the sequence of CYP707A gene family was highly conservative among different species. They contained the highly conserved cysteine residue, which was the putative heme iron ligand. Phylogenetic tree analysis showed that PpCYP707A1 protein was closer to StCYP707A1, AhCYP707A1, PvCYP707A1 and PvCYP707A2; PpCYP707A2 protein was closer to AtCYP707A2; PpCYP707A3 protein was closer to AtCYP707A4 and AhCYP707A2. Quantitative real-time PCR analysis demonstrated that PpCYP707A1, PpCYP707A2 and PpCYP707A3 was highly expressed in stem, leaf and flower, respectively, and PpCYP707As was higher expressed in mature tissues than in young tissues in general. PpCYP707As gene family members seemed playing overlapping role in controlling flower bud dormancy, and PpCYP707A2 could be induced by ABA and GA in peach, Finally, we obtained and purified the fused PpCYP707A1 and PpCYP707A2 protein by recombinant prokaryotic expression to further study on the functions of them.

Key words: peach; ABA 8’-hydroxylase; CYP707A; molecular cloning; expression analysis; bud dormancy; prokaryotic expression