欢迎访问作物学报，今天是
作物学报

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)

RichHTML

27

PDF

327

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。"

[1] 黄冰艳, 齐飞艳, 孙子淇, 苗利娟, 房元瑾, 郑峥, 石磊, 张忠信, 刘华, 董文召, 汤丰收, 张新友. 以分子标记辅助连续回交快速提高花生品种油酸含量及对其后代农艺性状的评价. 作物学报, 2019, 45:546-555.
Huang B Y, Qi F Y, Sun Z Q, Miao L J, Fang Y J, Zheng Z, Shi L, Zhang Z X, Liu H, Dong W Z, Tang F S, Zhang X Y. Acta Agron Sin, 2019, 45:546-555 (in Chinese with English abstract).
[2] Zhao Z, Shi A, Wang Q, Zhou J. High oleic acid peanut oil and extra virgin olive oil supplementation attenuate metabolic syndrome in rats by modulating the gut microbiota. Nutrients, 2019, 11:3005.
[3] Derbyshire E J. A review of the nutritional composition, organoleptic characteristics and biological effects of the high oleic peanut. Int J Food Sci Nutr, 2014, 65:1-10.
[4] Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browse J. ArabidopsisFAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell, 1994, 6:147-158.
[5] Jung S, Swift D, Sengoku E, Patel M, Teulé F, Powell G, Moore K, Abbott A. The high oleate trait in the cultivated peanut ( Arachis hypogaea L.): I. Isolation and characterization of two genes encoding microsomal oleoyl-PC desaturases. Mol Gen Genet, 2000, 5:796-805.
[6] Patel M, Jung S, Moore K, Powell G, Ainsworth C, Abbott A. High-oleate peanut mutants result from a MITE insertion into the FAD2 gene. Theor Appl Genet, 2004, 108:1492-1502.
[7] Sturtevant D, Horn P, Kennedy C, Hinze L, Percy R, Chapman K. Lipid metabolites in seeds of diverse Gossypium accessions: molecular identification of a high oleic mutant allele. Planta, 2017, 245:595-610.
[8] Long W, Hu M, Gao J, Chen S, Zhang J, Cheng L, Pu H. Identification and functional analysis of two new mutantBnFAD2 alleles that confer elevated oleic acid content in rapeseed. Front Genet, 2018, 9:399.
[9] Kulkarni K P, Patil G, Valliyodan B, Vuong T D, Shannon J G, Nguyen H T, Lee J D. Comparative genome analysis to identify SNPs associated with high oleic acid and elevated protein content in soybean. Genome, 2018, 61:217-222.
[10] Tian Y, Chen K, Li X, Zheng Y, Chen F. Design of high-oleic tobacco ( Nicotiana tabacum L.) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2-2. BMC Plant Biol, 2020, 20:233.
[11] Jung J H, Kim H, Go YS, Lee S B, Hur C G, Kim H U, Suh M C. Identification of functional BrFAD2-1 gene encoding microsomal delta-12 fatty acid desaturase from Brassica rapa and development ofBrassica napus containing high oleic acid contents. Plant Cell Rep, 2011, 30:1881-1892.
[12] Do P T, Nguyen C X, Bui H T, Tran L T N, Stacey G, Gillman J D, Zhang Z J, Stacey M G. Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean. BMC Plant Biol, 2019, 19:311.
[13] Chen Y, Fu M, Li H, Wang L, Liu R, Liu Z, Zhang X, Jin S. High oleic acid content, nontransgenic allotetraploid cotton ( Gossypium hirsutum L.) generated by knockout of GhFAD2 genes with CRISPR/Cas9 system. Plant Biotechnol J, 2021, 19:424-426.
[14] Suito T, Nagao K, Takeuchi K, Juni N, Hara Y, Umeda M. Functional expression of Δ12 fatty acid desaturase modulates thermoregulatory behaviour in Drosophila. Sci Rep, 2020, 10:11798.
[15] Welti R, Li W, Li M, Sang Y, Biesiada H, Zhou H E, Rajashekar C B, Williams T D, Wang X. Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing- induced lipid changes in Arabidopsis. J Biol Chem, 2002, 277:31994-32002.
[16] Heppard E P, Kinney A J, Stecca K L, Miao G H. Developmental and growth temperature regulation of two different microsomal omega-6 desaturase genes in soybeans. Plant Physiol, 1996, 110:311-319.
[17] Kargiotidou A, Deli D, Galanopoulou D, Tsaftaris A, Farmaki T. Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton ( Gossypium hirsutum). J Exp Bot, 2008, 59:2043-2056.
[18] Matteucci M, D’Angeli S, Errico S, Lamanna R, Perrotta G, Altamura M M. Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of Olea europaea L. genotypes with different cold hardiness. J Exp Bot, 2011, 62:3403-3420.
[19] D’Angeli S, Altamura M M. Unsaturated lipids change in olive tree drupe and seed during fruit development and in response to cold-stress and acclimation. Int J Mol Sci, 2016, 17:1889.
[20] Los D A, Ray M K, Murata N. Differences in the control of the temperature-dependent expression of four genes for desaturases in Synechocystis sp. PCC 6803. Mol Microbiol, 1997, 25:1167-1175.
[21] 薛晓梦, 李建国, 白冬梅, 晏立英, 万丽云, 康彦平, 淮东欣, 雷永, 廖伯寿. 花生FAD2基因家族表达分析及其对低温胁迫的响应. 作物学报, 2019, 45:1586-1594.
Xue X M, Li J G, Bai D M, Yan L Y, Wan L Y, Kang Y P, Huai D X, Lei Y, Liao B S. Expression profiles of FAD2 genes and their responses to cold stress in peanut. Acta Agron Sin, 2019, 45:1586-1594 (in Chinese with English abstract).
[22] 周婧雯, 丁玉娇, 曾晓蓉 Ganesh K J, 刘宝林, 韩颖颖. 生菜种子低温处理后耐冻性和脂肪酸合成代谢的关系研究. 亚热带植物科学, 2020, 49:9-14.
Zhou J W, Ding Y J, Zeng X R, Ganesh K J, Liu B L, Han Y Y. Relationship between freezing tolerance and fatty acid biosynthesis in lettuce seeds after low temperature treatment. Subtrop Plant Sci, 2020, 49:9-14 (in Chinese with English abstract).
[23] Miquel M F, Browse J A. High-oleate oilseeds fail to develop at low temperature. Plant Physiol, 1994, 106:421-427.
[24] Zhang J, Liu H, Sun J, Li B, Zhu Q, Chen S, Zhang H. Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth. PLoS One, 2012, 7:e30355.
[25] Feng T, Yang Y, Busta L, Cahoon E B, Wang H, Lyu S. FAD2 gene radiation and positive selection contributed to polyacetylene metabolism evolution in campanulids. Plant Physiol, 2019, 181:714-728.
[26] 张高华, 于树涛, 王鹤, 王旭达. 高油酸花生发芽期低温胁迫转录组及差异表达基因分析. 遗传, 2019, 41:1059-1073.
Zhang G H, Yu S T, Wang H, Wang X D. Transcriptome profiling of high oleic peanut under low temperatureduring germination. Hereditas, 2019, 41:1059-1073 (in Chinese with English abstract).
[27] 万书波. 中国花生栽培学. 上海: 上海科学技术出版社, 2003. pp 16-17.
Wan S B. Peanut Cultivation in China. Shanghai: Shanghai Scientific and Technical publishers, 2003. pp 16-17(in Chinese).
[28] Upchurch R G. Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress. Biotechnol Lett, 2008, 30:967-977.
[29] 于明洋, 孙明明, 郭悦, 姜平平, 雷永, 黄冰艳, 冯素萍, 郭宝珠, 隋炯明, 王晶珊, 乔利仙. 利用回交法快速选育高油酸花生新品系. 作物学报, 2017, 43:855-861.
Yu M Y, Sun M M, Guo Y, Jiang P P, Lei Y, Huang B Y, Feng S P, Guo B Z, Sui J M, Wang J S, Qiao L S. Breeding new peanut line with high oleic acid content using backcross method. Acta Agron Sin, 2017, 43:855-861 (in Chinese with English abstract).
[30] 张照华, 王志慧, 淮东欣, 谭家壮, 陈剑洪, 晏立英, 王晓军, 万丽云, 陈傲, 康彦平, 姜慧芳, 雷永, 廖伯寿. 利用回交和标记辅助选择快速培育高油酸花生品种及其评价. 中国农业科学, 2018, 51:1641-1652.
Zhang Z H, Wang Z H, Huai D X, Tan J Z, Chen J H, Yan L Y, Wang X J, Wan L Y, Chen A, Kang Y P, Jiang H F, Lei Y, Liao B S. Fast development of high oleate peanut cultivars by using marker-assisted backcrossing and their evaluation. Sci Agric Sin, 2018, 51:1641-1652 (in Chinese with English abstract).
[31] Wang Y, Zhang X, Zhao Y, Prakash C S, He G, Yin D. Insights into the novel members of the FAD2 gene family involved in high-oleate fluxes in peanut. Genome, 2015, 58:375-383.
[32] Liu Q, Brubaker C L, Green A G, Marshall D R, Sharp P J, Singh S P. Evolution of the FAD2-1 fatty acid desaturase 5' UTR intron and the molecular systematics of Gossypium (Malvaceae). Am J Bot, 2001, 88:92-102.
[33] Zhang D, Pirtle I L, Park S J, Nampaisansuk M, Neogi P, Wanjie S W, Pirtle R M, Chapman K D. Identification and expression of a new delta-12 fatty acid desaturase ( FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants. Plant Physiol Biochem, 2009, 47:462-471.
[34] Yuan S, Wu X, Liu Z, Luo H, Huang R. Abiotic stresses and phytohormones regulate expression of FAD2 gene inArabidopsis thaliana. J Integr Agric, 2012, 11:62-72.
[35] Kim M J, Kim H, Shin J S, Chung C H, Ohlrogge J B, Suh M C. Seed-specific expression of sesame microsomal oleic acid desaturase is controlled by combinatorial properties between negative cis-regulatory elements in the SeFAD2 promoter and enhancers in the 5'-UTR intron. Mol Genet Genomics, 2006, 276:351-368.
[36] Xiao G, Zhang Z Q, Yin C F, Liu R Y, Wu X M, Tan T L, Chen S Y, Lu C M, Guan C Y. Characterization of the promoter and 5'-UTR intron of oleic acid desaturase ( FAD2) gene in Brassica napus. Gene, 2014, 545:45-55.
[37] 刘睿洋, 刘芳, 张振乾, 官春云. 甘蓝型油菜BnFAD2-C5基因启动子及内含子在表达水平的功能分析. 作物学报, 2016, 42:1471-1478.
Liu R Y, Liu F, Zhang Z Q, Guan C Y. Functional analysis of BnFAD2-C5 promoter and intron at expression level in Brassica napus. Acta Agron Sin, 2016, 42:1471-1478 (in Chinese with English abstract).
[38] Harrison P M, Hegyi H, Balasubramanian S, Luscombe N M, Bertone P, Echols N, Johnson T, Gerstein M. Molecular fossils in the human genome: identification and analysis of the pseudogenes in chromosomes 21 and 22. Genome Res, 2002, 12:272-280.
[39] Xie J, Li Y, Liu X, Zhao Y, Li B, Ingvarsson P K, Zhang D. Evolutionary origins of pseudogenes and their association with regulatory sequences in plants. Plant Cell, 2019, 31:563-578.
[40] 肖钢, 张振乾, 邬贤梦, 谭太龙, 官春云. 六个甘蓝型油菜油酸脱氢酶(FAD2)假基因的克隆和分析. 作物学报, 2010, 36:435-441.
Xiao G, Zhang Z Q, Wu X M, Tan T L, Guan C Y. Cloning and characterization of six oleic acid desaturase pseudogenes of Brassica napus. Acta Agron Sin, 2010, 36:435-441 (in Chinese with English abstract).
[1] 杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487.
[2] 李海芬, 魏浩, 温世杰, 鲁清, 刘浩, 李少雄, 洪彦彬, 陈小平, 梁炫强. 花生电压依赖性阴离子通道基因(AhVDAC)的克隆及在果针向地性反应中表达分析[J]. 作物学报, 2022, 48(6): 1558-1565.
[3] 刘嘉欣, 兰玉, 徐倩玉, 李红叶, 周新宇, 赵璇, 甘毅, 刘宏波, 郑月萍, 詹仪花, 张刚, 郑志富. 耐三唑并嘧啶类除草剂花生种质创制与鉴定[J]. 作物学报, 2022, 48(4): 1027-1034.
[4] 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596.
[5] 丁红, 徐扬, 张冠初, 秦斐斐, 戴良香, 张智猛. 不同生育期干旱与氮肥施用对花生氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 695-703.
[6] 黄莉, 陈玉宁, 罗怀勇, 周小静, 刘念, 陈伟刚, 雷永, 廖伯寿, 姜慧芳. 花生种子大小相关性状QTL定位研究进展[J]. 作物学报, 2022, 48(2): 280-291.
[7] 汪颖, 高芳, 刘兆新, 赵继浩, 赖华江, 潘小怡, 毕晨, 李向东, 杨东清. 利用WGCNA鉴定花生主茎生长基因共表达模块[J]. 作物学报, 2021, 47(9): 1639-1653.
[8] 王建国, 张佳蕾, 郭峰, 唐朝辉, 杨莎, 彭振英, 孟静静, 崔利, 李新国, 万书波. 钙与氮肥互作对花生干物质和氮素积累分配及产量的影响[J]. 作物学报, 2021, 47(9): 1666-1679.
[9] 高芳, 刘兆新, 赵继浩, 汪颖, 潘小怡, 赖华江, 李向东, 杨东清. 北方主栽花生品种的源库特征及其分类[J]. 作物学报, 2021, 47(9): 1712-1723.
[10] 张鹤, 蒋春姬, 殷冬梅, 董佳乐, 任婧瑶, 赵新华, 钟超, 王晓光, 于海秋. 花生耐冷综合评价体系构建及耐冷种质筛选[J]. 作物学报, 2021, 47(9): 1753-1767.
[11] 薛晓梦, 吴洁, 王欣, 白冬梅, 胡美玲, 晏立英, 陈玉宁, 康彦平, 王志慧, 淮东欣, 雷永, 廖伯寿. 低温胁迫对普通和高油酸花生种子萌发的影响[J]. 作物学报, 2021, 47(9): 1768-1778.
[12] 郝西, 崔亚男, 张俊, 刘娟, 臧秀旺, 高伟, 刘兵, 董文召, 汤丰收. 过氧化氢浸种对花生种子发芽及生理代谢的影响[J]. 作物学报, 2021, 47(9): 1834-1840.
[13] 张旺, 冼俊霖, 孙超, 王春明, 石丽, 于为常. CRISPR/Cas9编辑花生FAD2基因研究[J]. 作物学报, 2021, 47(8): 1481-1490.
[14] 戴良香, 徐扬, 张冠初, 史晓龙, 秦斐斐, 丁红, 张智猛. 花生根际土壤细菌群落多样性对盐胁迫的响应[J]. 作物学报, 2021, 47(8): 1581-1592.
[15] 黄冰艳, 孙子淇, 刘华, 房元瑾, 石磊, 苗利娟, 张毛宁, 张忠信, 徐静, 张梦圆, 董文召, 张新友. 花生巢式群体的脂肪含量遗传分析[J]. 作物学报, 2021, 47(6): 1100-1108.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2