《植物生理学报》 2017, 53(1): 79-88

星油藤关键酶基因PvFAD3启动子的克隆及表达激活分析

廖晓佳^1,3, 王晓娟², 王彬^1,3, 刘爱忠^1,*

¹中国科学院昆明植物研究所, 昆明650201; ²周口师范学院生命科学系, 河南周口466000; ³中国科学院大学, 北京100049

通信作者：刘爱忠；E-mail: liuaizhong@mail.kib.ac.cn

摘　要：

α-亚麻油酸(α-linolenic acid, ALA)对防治脂类代谢失调而引起的肥胖、心血管疾病、高血脂、高血压等慢性疾病具有重要的生理功能。然而, 大部分油料作物种子ALA的含量都很低, 提高种子ALA的含量一直是油料作物品质改良的重点内容之一。星油藤(Plukenetia volubilis)种子油中富含ALA, 是一种具有重要开发前景的油料植物, 解析其种子高效累积ALA的分子机理不仅能为星油藤的遗传改良提供理论依据, 而且能够为提高油料作物种子ALA含量的遗传育种提供理论参考。前期的研究发现星油藤种子高效累积ALA主要是由于关键酶基因PvFAD3高度表达的结果。为了进一步理解星油藤种子在发育过程中PvFAD3的被调控机理, 本研究利用染色体步移法克隆了PvFAD3的启动子, 并且利用生物信息学分析、鉴别了启动子潜在的关键调控元件, 并重点围绕可能调控PvFAD3基因表达的bZIP124转录因子, 通过酵母单杂交和烟草叶片启动子激活实验, 验证了PvFAD3的表达受bZIP124转录因子的调控。该研究为理解植物种子高效累积ALA的分子机理提供了新的证据。

关键词：星油藤; α-亚麻油酸; bZIP转录因子; FAD3基因; 启动子区; 转录激活

收稿：2016-11-07   修定：2016-12-19

资助：国家自然科学基金(31571709)和云南省应用基础自然科学基金(2014FB172)。

Cloning of the promoter of the key enzyme gene PvFAD3 from Sacha Inchi and its transcriptional activation analyses

LIAO Xiao-Jia^1,3, WANG Xiao-Juan², WANG Bin^1,3, LIU Ai-Zhong^1,*

¹Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China; ²Department of Life Sciences, Zhoukou Normal University, Zhoukou, Henan 466000, China; ³University of Chinese Academy of Sciences, Beijing 100049, China

Corresponding author: LIU Ai-Zhong; E-mail: liuaizhong@mail.kib.ac.cn

Abstract:

The α-linolenic acid (ALA) is essential in diet because of its functions in taking precautions against disturbance of lipid metabolism. The content of ALA, however, is low in most of oilseeds. Increasing the ALA content is critical to enhance the oil quality for genetic improvement and breeding in oilseed crops. Still, the molecular mechanism of ALA biosynthesis in oilseeds remains uncertain. Sacha Inchi (Plukenetia volubilis) is a potential oilseed crop because its seed oil is rich in ALA (ca. 50%). Dissecting the molecular mechanism underlying ALA biosynthesis in Sacha Inchi seeds would provide the information serving for genetic improvement and breeding toward enhancing the oil quality in oilseed crops. Our previous study has revealed that the high expression of key enzyme gene PvFAD3 encoding the desaturase which makes the formation of ALA is responsible for the high content of ALA in the developing seeds of Sacha Inchi. Further, in order to investigate how PvFAD3 is regulated in the developing seeds of Sacha Inchi, we cloned the promoter region of PvFAD3 gene using the Tail-PCR technology, and analyzed the main cis-elements within the promoter by bioinformatics methods in this study. We identified the transcription factor bZIP124, which might be a key factor to regulate the expression of PvFAD3 gene. Finally, we experimentally confirmed that the transcriptional activation of PvFAD3 using the yeast one hybrid technique and the transcriptional activation experiment in tobacco leaf. This study would add novel insights in understanding the regulatory mechanisms of ALA biosynthesis in Sachi Inchi seeds.

Key words: Sacha Inchi; α-linolenic acid (ALA); bZIP transcription factor; FAD3 gene; promoter region; transcriptional activation