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作物学报 ›› 2021, Vol. 47 ›› Issue (9): 1703-1711.doi: 10.3724/SP.J.1006.2021.04247

• 研究论文 • 上一篇    下一篇

花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答

石磊1,2(), 苗利娟1,2, 黄冰艳1,2, 高伟3, 张忠信1,2, 齐飞艳1,2, 刘娟3, 董文召1,2, 张新友1,2,*()   

  1. 1河南省农业科学院河南省作物分子育种研究院 / 农业农村部黄淮海油料作物重点实验室 / 河南省油料作物遗传改良重点实验室 / 花生遗传改良国家地方联合工程实验室, 河南郑州 450002
    2河南生物育种中心有限公司, 河南郑州 450002
    3河南省农业科学院经济作物研究所, 河南郑州 450002
  • 收稿日期:2020-11-17 接受日期:2021-03-19 出版日期:2021-09-12 网络出版日期:2021-03-31
  • 通讯作者: 张新友
  • 作者简介:E-mail: leis100@163.com
  • 基金资助:
    国家重点研发计划项目“大田经济作物优质丰产的生理基础与调控”(2018YFD1000900);国家现代农业产业技术体系建设专项(CARS-13);河南省现代农业产业技术体系项目(S2012-5)

Characterization of the promoter and 5'-UTR intron in AhFAD2-1 genes from peanut and their responses to cold stress

SHI Lei1,2(), MIAO Li-Juan1,2, HUANG Bing-Yan1,2, GAO Wei3, ZHANG Zong-Xin1,2, QI Fei-Yan1,2, LIU Juan3, DONG Wen-Zhao1,2, ZHANG Xin-You1,2,*()   

  1. 1Henan Academy of Crops Molecular Breeding, Henan Academy of Agricultural Sciences / Key Laboratory of Oil Crops in Huanghuaihai Plains, Ministry of Agriculture and Rural Affairs / Henan Provincial Key Laboratory for Oil Crops Improvement / National and Provincial Joint Engineering Laboratory for Peanut Genetic Improvement, Zhengzhou 450002, Henan, China
    2Henan Biological Breeding Center Co., Ltd., Zhengzhou 450002, Henan, China
    3Industrial Crops Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
  • Received:2020-11-17 Accepted:2021-03-19 Published:2021-09-12 Published online:2021-03-31
  • Contact: ZHANG Xin-You
  • Supported by:
    National Key Research and Development Program of China “Physiological Basis and Agronomic Management for High-quality and High-yield of Field Cash Crops”(2018YFD1000900);China Agriculture Research System(CARS-13);Henan Province Agriculture Research System(S2012-5)

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摘要：

Δ12-脂肪酸脱氢酶基因(FAD2)催化油酸生成亚油酸, 是决定油酸亚油酸比值的关键基因。高油酸花生对低温更加敏感, 暗示FAD2在低温响应中发挥作用。为探索花生FAD2的低温胁迫应答, 本研究分析了花生FAD2-1A/B的基因结构, 从普通油酸花生品种豫花9326中克隆了FAD2-1A/B启动子和内含子, 并通过转化拟南芥验证了功能及其对冷胁迫的应答。结果表明, AhFAD2-1A/B包含2个外显子和1个位于5'-UTR的内含子; AhFAD2-1A/B基因启动子功能较弱, 仅AhFAD2-1B启动子在子叶期幼苗的子叶叶尖中观察到蓝色; AhFAD2-1假基因5'侧翼序列具有启动子活性, 可调控基因在子叶、下胚轴、种子中表达。AhFAD2-1内含子序列具有启动子的功能, 驱动基因在幼苗下胚轴及子叶中表达, 同时还有提高基因表达效率和扩大表达范围的功能, 是AhFAD2-1基因表达调控必需元件; 包含5'-UTR内含子的AhFAD2-1A/B启动子的调控序列功能受低温胁迫抑制。

关键词: 花生, Δ12-脂肪酸脱氢酶基因(FAD2), 启动子, 内含子介导增强, 冷胁迫, GUS报告基因

Abstract:

Delta-12 fatty acid desaturase 2 (FAD2) catalyzes the conversion of oleic acid to linoleic acid and is the determination of the level of oleic to linoleic acid ratio (O/L) in peanut oil. Peanuts with high oleic acid content are more susceptible to cold stress than those with normal oleic acid content, suggesting that FAD2 plays important roles in response to cold stress. To explore the role ofFAD2s during the process of cold stress acclimation in peanut, the genomic structures of AhFAD2-1A/Bwere determined; the function of promoters, intron of AhFAD2-1A/Band their response to cold stress were characterized by using β-glucuronidase (GUS) gene reporter system in transgenic Arabidopsis. The results were as follows: AhFAD2-1A/B genes consisted of two exons and one intron within their 5'-UTR; promoter ofAhFAD2-1A was too weak to be detected and the promoter of AhFAD2-1B poorly activated the expression level of GUS in cotyledon tip of seedlings; the promoter of AhFAD2-1pseudogene activated GUS expression limited to cotyledon, hypocotyl, and seed. The intron of AhFAD2-1B demonstrated promoter-like activity which was restricted in cotyledon and hypocotyl, and not only enhanced the gene expression efficiency but also expanded gene expression range. Intron-mediated enhancement was an essential aspect of AhFAD2-1expression. Activities of 5'-flanking region of AhFAD2-1A/B were repressed by the cold stress.

Key words: peanut (Arachis hypogaea L.), Δ12-fatty acid desaturase gene (FAD2), promoter, intron-mediated enhancement, cold stress, GUS reporter gene

表1

本研究所用引物及其序列"

目的片段
Target fragment 		上游引物
Forward primer (5°-3°) 		下游引物
Reverse primer (5°-3°)
PAhFAD2-1A+intron ACGCGTCGA (Sal I) CTTCCGCCAGAATGAGGAACTAAA TCACAG GACATCTAGA (Xba I) GTTGTGTTGTTAAAGCT CCTGTTACCAATG
PAhFAD2-1B+intron CGGGGTACC (Kpn I) ATGTTTCATAGAATTTAAGCTCAGA CACG
PAhFAD2-1B(pseudo) ACGCGTCGA (Sal I) CACCAAGTAGCTTCTCAATGGCTCA GATTCG
AhFAD2-1B-intron ACGCGTCGA (Sal I) CATAGGAGAAGCACTCACTTCTCTT CTCTC
AhFAD2-1B-intron681 ACGCGTCGA (Sal I) CATAATGGCTTCTGGGCCCTCAC
AhFAD2-1B-intron363 ACGCGTCGA (Sal I) CATAATGGCTTCTGGGCCCTCAC GACATCTAGA (Xba I) TATCATGCCAAGTGACT AGTATGA
PAhFAD2-1A ACGCGTCGA (Sal I) CTTCCGCCAGAATGAGGAACTAAA TCACAG GACATCTAGA (Xba I) GATGAATCTCGCAGCCA CGTTT
PAhFAD2-1B CGGGGTACC (Kpn I) ATGTTTCATAGAATTTAAGCTCAGA CACG

图1

FAD2-1A/B/pseudo基因结构及启动子比较 TSS: 转录起始位点。"

图2

AhFAD2A/B/pseudo启动子和内含子转基因拟南芥的GUS组织化学染色 "

图3

转PAhFAD2-1A+intron拟南芥低温胁迫条件下的GUS组织化学染色 A: 转PAhFAD2-1A+intron拟南芥; B: 转CaMV35S拟南芥。1-day: 处理1 d; 2-day: 处理2 d; 4-day: 处理4 d。 "

图4

转PAhFAD2-1B+intron拟南芥低温胁迫条件下的GUS组织化学染色 1-day: 处理1 d; 2-day: 处理2 d; 4-day: 处理4 d。"

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