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作物学报

作物学报 ›› 2026, Vol. 52 ›› Issue (4): 1268-1276.doi: 10.3724/SP.J.1006.2026.54079

• 研究简报 • 上一篇    

甘薯IbOPR2基因启动子克隆及调控因子的筛选

王懿涵1,2(), 李富昌1, 刘意1,2,*(), 朱国鹏1,2,*()   

  1. 1海南大学南繁学院 / 海南大学三亚南繁研究院, 海南三亚572025
    2海南省热带园艺作物品质调控重点实验室 / 海南大学热带农林学院, 海南海口570228
  • 收稿日期:2025-07-02 接受日期:2025-11-19 出版日期:2026-04-12 网络出版日期:2025-11-26
  • 通讯作者: *刘意, E-mail: 996178@hainanu.edu.cn; 朱国鹏, E-mail: zhuguopeng@hainanu.edu.cn
  • 作者简介:E-mail: 23210902000002@hainanu.edu.cn
  • 基金资助:
    “崖州湾”菁英人才基金项目(SKJC-JYRC-2024-22);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-10-Sweetpotato)

Cloning of the IbOPR2 gene promoter and identification of regulatory factors in sweetpotato

Wang Yi-Han1,2(), Li Fu-Chang1, Liu Yi1,2,*(), Zhu Guo-Peng1,2,*()   

  1. 1School of Breeding and Multiplication / Sanya Institute of Breeding and Multiplication, Hainan University, Sanya 572025, Hainan, China
    2Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province / Tropical Agriculture and forestry College, Hainan University, Haikou 570228, Hainan, China
  • Received:2025-07-02 Accepted:2025-11-19 Published:2026-04-12 Published online:2025-11-26
  • Contact: *E-mail: 996178@hainanu.edu.cn; E-mail: zhuguopeng@hainanu.edu.cn
  • Supported by:
    “Yazhou Bay” Elite Talent Project Fund(SKJC-JYRC-2024-22);China Agriculture Research System of MOF and MARA(CARS-10-Sweetpotato)

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摘要：

茉莉酸类物质(JA)作为植物内源激素, 广泛参与植物耐盐胁迫过程, 12-氧-植物二烯酸还原酶(OPR)作为JA合成关键酶, 其表达调控机制尚不明确。为筛选与鉴定调控IbOPR2基因表达的转录因子, 本研究以甘薯耐盐品系‘海大7791’为材料, 克隆IbOPR2基因启动子序列。采用生物信息学方法分析其顺式作用元件, 并通过转录激活试验检测其启动子活性。利用酵母单杂交试验筛选其互作转录因子, 并通过酵母单杂交点对点验证试验确认结合特异性。结果表明, 克隆的IbOPR2基因上游1964 bp启动子序列含有MYBHv1、MYB和WRKY识别位点元件、MYB和MYC等胁迫响应元件、MeJA激素应答元件, 并且启动子具有显著转录激活能力。以MeJA应答元件和MYC元件作为诱饵的酵母单杂交试验, 共筛选到5个候选转录因子, 分别为IbPHL7、IbZFP16、IbSKIP11、IbTGA2和IbERF2。随后酵母单杂交点对点试验发现, IbPHL7、IbZFP16和IbSKIP11均能与IbOPR2启动子元件特异性结合。双荧光素酶报告基因系统试验结果表明, IbPHL7、IbZFP16和IbSKIP11可以激活IbOPR2启动子的转录活性。qRT-PCR分析结果表明, IbPHL7、IbZFP16和IbSKIP11均响应甘薯的盐胁迫。以上结果表明, 它们可能作为IbOPR2上游调控因子, 调控IbOPR2基因表达。本研究首次筛选了甘薯IbOPR2上游调控因子, 为深入解析IbOPR2响应盐胁迫的表达调控机制奠定了基础。

关键词: 甘薯, IbOPR2, 酵母单杂, 转录因子, 启动子

Abstract:

Jasmonic acids (JAs), as endogenous plant hormones, play a crucial role in the plant response to salt stress. 12-oxophytodienoate reductase (OPR), a key enzyme in JA biosynthesis, remains poorly understood in terms of its expression regulation. To identify transcription factors involved in regulating IbOPR2 expression, this study used the salt-tolerant sweetpotato cultivar ‘Haida 7791’ to clone the promoter sequence of the IbOPR2 gene. Bioinformatics analysis revealed various cis-acting elements, and transcriptional activation assays confirmed promoter activity. Yeast one-hybrid (Y1H) screening was conducted to identify transcription factors interacting with the promoter, and point-to-point Y1H assays were used to validate specific binding. A 1964 bp promoter sequence upstream of IbOPR2 was successfully cloned, containing recognition motifs such as MYBHv1, MYB, and WRKY, along with stress-responsive elements (MYB, MYC) and MeJA-responsive elements, indicating strong transcriptional activation potential. Five candidate transcription factors—PHL7, ZFP16, SKIP11, TGA2, and ERF2—were identified using MYC and MeJA response elements as bait in Y1H assays. Follow-up point-to-point verification confirmed that IbPHL7, IbZFP16, and IbSKIP11 specifically bind to the IbOPR2 promoter. Dual-luciferase reporter assays further demonstrated that these three transcription factors could activate the IbOPR2 promoter. Moreover, qRT-PCR analysis showed that the expression of IbPHL7, IbZFP16, and IbSKIP11 is responsive to salt stress in sweetpotato. Taken together, these results suggest that these transcription factors may function as upstream regulators of IbOPR2, modulating its expression under salt stress conditions. This study represents the first identification of transcriptional regulators of sweetpotato IbOPR2, providing a foundation for further investigation into its regulatory mechanisms in response to salt stress.

Key words: sweetpotato, IbOPR2, yeast one-hybrid, transcription factor, promoter

附表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 目的
Purpose
IbOPR2-pro-F GTGTGGTAGGACGACGTTGTAG IbOPR2启动子克隆
IbOPR2 promoter cloning
IbOPR2- pro-R TTGGGTAGCCCTCTGTGAATAG
motif-F gcttgaattcgagctcggtaccGATCGTCAACTCAGCAATTGTTCGATCGTCAACTCAGCAATTGTTCGATCGTCAACTCAGCAATTGTTCctcgaggcatgtgctctgtat Motif截断IbOPR2启动子扩增
Motif truncates IbOPR2 promoter amplification
motif-R atacagagcacatgcctcgagGAACAATTGCTGAGTTGACGATCGAACAATTGCTGAGTTGACGATCGAACAATTGCTGAGTTGACGATCggtaccgagctcgaattcaagc
GUS-F gccaagctttctagaggatccGTGTGGTAGGACGACGTTGTAG IbOPR2启动子活性检测载体构建
Construction of the IbOPR2 promoter activity detection vector
GUS-R gcccttgctcaccatggtaccTTGGGTAGCCCTCTGTGAATAG
PHL7-CDS-F ATGTACCAACCAAAAGCTGTTCT PHL7 CDS序列扩增
PHL7 CDS sequence amplification
PHL7-CDS-R TCAGCCAAGTAACTTAGTAAGCA
ZFP16-CDS-F ATGGGGACGCCGGAA ZFP16 CDS序列扩增
ZFP16 CDS sequence amplification
ZFP16-CDS-R CTATACCTTTGTGAAGTTCCTGTG
SKIP11-CDS-F ATGGTGAACCCTGAAGGGATGTT SKIP11 CDS序列扩增
SKIP11 CDS sequence amplificatio
SKIP11-CDS-R TTAGCAACCCATCACCGCA
TAG-CDS-F ATGGCCGGGGGAGGAGGG TAG CDS序列扩增
TAG CDS sequence amplification
TAG-CDS-R TCAGAAGTTTGCGAATTGGTTG
ERF2-CDS-F ATGTGCGGTGGTGCTATAATCTC ERF2 CDS序列扩增
ERF2 CDS sequence amplification
ERF2-CDS-R CTAGAAGACACCCCCCATTAAAGA
pGADT7-PHL7-F ggccagtgaattccacccgggATGTACCAACCAAAAGCTGTTCTTAG 酵母单杂交点对点验证载体构建
Construction of yeast single-hybrid point-to-point validation vectors
pGADT7-PHL7-R acgattcatctgcagctcgagTCAGCCAAGTAACTTAGTAAGCATTG
pGADT7-ZFP16-F ggccagtgaattccacccgggATGGGGGCGCCGGAACTT 酵母单杂交点对点验证载体构建
Construction of yeast single-hybrid point-to-point validation vectors
pGADT7-ZFP16-R acgattcatctgcagctcgagCTATACCTTTGTGAAGTTCCTGTGCT
pGADT7-TAG-F ggccagtgaattccacccgggATGGCCGGGGGAGGAGGG 酵母单杂交点对点验证载体构建
Construction of yeast single-hybrid point-to-point validation vectors
pGADT7-TAG-R acgattcatctgcagctcgagTCAGAAGTTTGCGAATTGGTTG
pGADT7-ERF2-F ggccagtgaattccacccgggATGTGCGGTGGTGCTATAATCTC 酵母单杂交点对点验证载体构建
Construction of yeast single-hybrid point-to-point validation vectors
pGADT7-ERF2-R acgattcatctgcagctcgagCTAGAAGACACCCCCCATTAAAGA
pGATD7-SKIP11-F ggccagtgaattccacccgggATGGTGAACCCTGAAGGGATG 酵母单杂交点对点验证载体构建
Construction of yeast single-hybrid point-to-point validation vectors
pGATD7-SKIP11-R cagctcgagctcgatggatccTTAGCAACCCATCACCGCA
pGreen-0800- proIbOPR2-F atcgaattcctgcagcccgggACATGCTGAAGTGACTGGTTGGC 双荧光素酶活性检测载体构建
Construction of dual-luciferase activity detection vectors
pGreen-0800- proIbOPR2-R cgctctagaactagtggatccGGTGATGAACAATTGCTGAGTTGA
pGreen-62SK-IbPHL7-F cgctctagaactagtggatccATGTACCAACCAAAAGCTGTTCTTAG 双荧光素酶活性检测载体构建
Construction of dual-luciferase activity detection vectors
pGreen-62SK-IbPHL7-R atcgaattcctgcagcccgggTCAGCCAAGTAACTTAGTAAGCATTG
pGreen-62SK-IbZFP16-F agaactagtggatcccccgggATGGGGACGCCGGAACTT 双荧光素酶活性检测载体构建
Construction of dual-luciferase activity detection vectors
pGreen-62SK-IbZFP16-R tcagcgtaccgaattggtaccCTATACCTTTGTGAAGTTCCTGTGCT
pGreen-62SK-SKIP11-F cgctctagaactagtggatccATGGTGAACCCTGAAGGGATG 双荧光素酶活性检测载体构建
Construction of dual-luciferase activity detection vectors
pGreen-62SK-SKIP11-R atcgaattcctgcagcccgggTTAGCAACCCATCACCGCA
Actin-F AGCAGCATGAAGATTAAGGTTGTAGCAC 荧光定量扩增
Fluorescence quantitative amplification
Actin-R TGGAAAATTAGAAGCACTTCCTGTGAAC
qPCR-IbPHL7F TGTGCAAGGGCTGACAATTTA 荧光定量扩增
Fluorescence quantitative amplification
qPCR-IbPHL7R TGCATCGGGAAGGTATTTGG
qPCR-IbZFP16F TGCCGGCAGATTGATTTCCT 荧光定量扩增
Fluorescence quantitative amplification
qPCR-IbZFP16R CACAGAGCGGACAGACAACT
qPCR-IbSKIP11F GAATGCAGATGAAAGAGCCACG 荧光定量扩增
Fluorescence quantitative amplification
qPCR-IbSKIP11R CAACCGCCAGCAAGAATAGC

图1

甘薯IbOPR2基因的启动子克隆 1 : IbOPR2的扩增产物。"

附表2

基因序列详情"

基因名称
Gene name		 基因序列
Gene sequence (5°-3°)
IbOPR2-PRO GTGTGGTAGGACGACGTTGTAGCGTCAGTCGCCCAAGATAAAGTCCGGCGAACTAGAATCGAGACCGAACACTTTGGTATTTTATCGGGAGTGTGGGCATAAACTTTTCAAGAAGATATGTTTTTCGCGAAAGAGAGGGGAGAAAAACAACAAAATTTTTTATTGTTCAAAATCAACAGCTCCAAATTCTGCCCAACATCTAGGTGTTTATATAGGAGATTGGAATTAAATTGCACTAATCAATGACACATAAATCCAATTTAATAATTAGATTATAACGTTTGAAACAGTCATATTTATTAATTATTATTAAATAATGTTTGCGGTTACGAAATTAAGTACAATCAATTCATACTGACCAAATTACATGTGCTTGACACGCTCGAAAGGTAAATTAACTTTTCGAAATTCACCAAATAGTCACAACCCCAAAAGTGCTTTAATTCATGTGCTAACAAAGCACTATTCGAAGTGCTGCAAATTAGCACATCACAATGCTAAACAAAATTAGCACCTTCCGTTAGTGCTACAAAAGTACTTCAACCTTTCATAATTTTGACTTCTCTAAAATAATTAAGTTCATTTTGAGAACCAATTTCTCTTATCCACCCATTATTCATTTTGAGCATACAATATATCAATTCTTTATGTTACTACGAAAAGTGTGAATTCACTTTGACCCAATCCAAATTGAAAGTAGACCCACATATATTTGTATCACAAAATAGCCACTTAAATGTCATTTTTATGTTGTCAAGACCTTGTGATCTAGTGGCACCCGGTTGCACTCCCATATGGAAAGGGGTGGGCTCGAGTCTCAGCCTCGCGTCATGTGCGTTGTCAGTGTTTCTTTAAAGAAATAGTTCCCTCCCAAGAAACTTTATCCCCTCCTCGACAAAAGCAAAAATTGACTATCCTGAAAAGAATGCATAAATAGGTCAAACCAAATCTCATATTTAAATGTCACAAGTTCAATAATTATCAATACCGTCTTAATCAAATTTATCGTACTATTACACGCAACAAAGTTCATTGCACATGGTTTTCAAAAAAAAAAAAAAAAAAAGTTCATTGCACATATAAAGGAAGAGGACGAAATAGCATCCACAATGACATACACTCCTACTTGGTTCATCTATTTTTATTTTTGGAGTATTACGTATTTCAATTAAATAAATATCCAATAAAAAAAAAACTAAAAATATGACTACTACTCTACTTATTACTTTTTTCCCAAGTGTGCCCCATGATCCACTTTATAAAATAGGCCACTATTCTCACTATATAAAGTTCATTTTGAATACATTAAATATTACTTTTTAAAATTTTATTTTTATTTATTAAAAAGTTCATTTATGAACATTGGATACATTAAAAGTTCAAATGACATGTAGTGACTCTCCCATATGGGAGGTCATGAGATCGAGTCTCAGTGGAGACAGTATTAACTCTCTTTGTGCTTCAACAGGAAAGGGTGATCATGGTCTCTGTCTCCGTCGCCTCGGAGCTCTACTACTGCATTATTGACATGCTATTGCAAGTCACTAAATTAATATGTTGATATGTTTAATTACAACTTTCAAAAGTTCAGGACCTAATTGACTCTTTGGATAAAGTTGGAAAGTTAATTTGATCATTTTCCTACTTAAAATGAACACTATATATAATTATTTGTAATATTTTCAACGGCCAAGTTAATTTGATCATTTTCCTACTTAAAATGAACACTATAATTATTTGTAATATTTTCAACGGCCAAATATCTTATTCCATTCTCATTTTCCGCACACCATTTCTGTGGAAATTGTGCGTCAATTCTGACCTTTTTGACTCTCCTCCATTCAGCAAAGTAGTGCATCATACTTTCTCAACTTTATATTCTCTTCAACTTCTCTTCCTCCCCATCTCTCTCCCTTTCTCTACAGATTCACTTCTGATCGTCAACTCAGCAATTGTTCATCACCATGGCTAGAACTTCGTCGCTGGATCAAGGCTCAACGCCAAGCCTCTTTTCCCCCTACAAGATGGGCAGGTTCAACCTCTCCCACAGGTCAGTTTTGCTTCAAAACGAAGTGGGTACTTCGTTTGTTTCACGATCCTTGACAAAACAAGAATGGGTATTTTTGTATCTGCCCTGAATCTCTAGATCTCTCTGCATTTTGGTAAAAAAATCTCAAAAAGTTGGAGACTTTAGGCTGATTATTAGTGATTACAAATAACCCTTGAAATTTCTGTGGTATACTATTGCAGGGTAGTATTGGCTCCATGTACAAGGCGTAGAGCAATAAATGGAATTCCTCAGCCGGCCCTGGCGGAGTACTATTCACAGAGGGCTACCCAA
IbPHL7 ATGTACCAACCAAAAGCTGTTCTTAGTTCAAGCTTAGTACCCAACAAGGCAATAGTCAGCCAGCAGTCATTAGACTTTAGTGCCAGCACAATGGAACCAATCAATGGGGGTAACAATCTCAACAACAATCCTAGTCTTGCTTCAAAACAACGCCTGCGTTGGTCACATGAGCTTCATGAAAGATTTGTTGATGCAGTTGCACAACTTGGTGGCCCAGATAGGGCTACTCCTAAAGGTGTTTTGAGAGTCATGGGTGTGCAAGGGCTGACAATTTATCATGTAAAAAGCCACTTACAGAAATATCGACTTGCCAAATACCTTCCCGATGCATCATCTGATGGAAAAAAACCCGACAATAAAGATTCAAGTGATATAATCTCCGGCCTAGATGATTCATCTGGAATGCAAATAACTGAGGCACTAAAATTGCAGATGGAGGTCCAAAAGCGACTACACGAGCAACTAGAGGTTCAGCGCCAGCTACAGCTAAGGATAGAGGCCCAAGGCAAGTATTTGAAGAAGATAATAGAAGAACAACAGCAACTCGGTGCTGTTTCAGAAATGCCAGAGTCCGGGGCTACGTCTGCATCAGCAGGCGAGAATGCCCCGGTATCTGATAACAGTACAAATCTAGTGACGCCTGTACCTGCATCTGAACCTCCTCATACTGAAAACCCTGCAAACGAATGCAATCCAGTTAAGGGCATTTCGCTTAACGCCTCGCCATCTTCTCATAACGAGGCTCTTTCACCAGATTCCAGCTGCCATGCAGCTTCTGCAGTCGAGAGGCCATCAAAGAAACAAAAAGTTAGTGCAGAAGCTGCTGCGTTTACCCAACCCGACGCAATGCTTACTAAGTTACTTGGCTGA
IbZFP16 ATGGGGACGCCGGAACTTCCAAACCTGGGAAAGCACTGCTCCGTCGACGATTGCCGGCAGATTGATTTCCTGCCGTTCACCTGCGATTGCTGTCAGAAGGTCTTTTGTCTAGAGCATCGAAGCTACTTGAGGCATCACTGTCCAACAGCCAATAAAAACGATGTTACGGTAGTTGTCTGTCCGCTCTGTGCTAAAGGTGTTCACTTAATTCCAGATGAAGACCCAAATGTGACCTGGGAGTTGCATGTGAACATTGATTGTGATCCGTCAAACTATGAAAAATCCACGAAGAAGAGGAAGTGTCCCGTGCCTGGTTGCAAAGAACTCCTGACATTCTCAAACACAATTAAGTGTCGAGATTGTAATGCAGATCACTGTTTGAAACACCGGTTTGGACCTGATCACAAATGTCCTGGACCTAGGAAACCCGAAACGTCTTTCCCATTCATGGGCTATTTGAGTAAGAGTAGAAAGGACGAGACAAAGCATGCCCCCGCTACTTCTTCCTCGAGCTGGACCACCACTTTCCTCGGGGCAGCATCTTCTGTTCGGGCAAAAGCTGGAGCTGGAATGGCAAAACTGAGCACCGAGTTCAATCAAGCACTGCAGAAAGGAAAAGACCGGGACAGCCTTAACAGCAGAGCCACAAGACAAGGTGGGATTACGAGTGGGAGTACCGGTGGAAATGTAGAACAATGCCCACAGTGCAACATGCGATTCTCCTCGGTTGTGGACCTCGTGGATCATGTGCAGAACGTCCACGAAAGGAAAAGCAAGAGCGTTACAAATTCGACAGTTGACATCTGCCCGAGATGCAGTAAAGGTTTTCACGATCCAGTTTCCCTCGTGGAACATGTCGAAAGGGAGCACAGGAACTTCACAAAGGTATAG
IbSKIP11 ATGGTGAACCCTGAAGGGATGTTGGAAGAGCGATCTTGTTTGGTATCGAGGAACTACCGGAGAGAGCATACCGGGACTTGTATGAATTACTGGATTGATGTGAATGGCAAAAGGCCATTCGAAGATGACAAGGAGAATGAACTGGTTCCTAGCAAGTTTGCAAGGCAATCTGACGGTGGTTGTGATAGAGTTGATACAGCTCTAACCCTAGGTGTTTCTTCAGCGTCCTGTGCTGAAGAAGTTGGTGATCGACCTGCTGATGGAGTGGAAACGAGTCTAACACTAGGCGGTGATATGTTGGCCCCTATTGGCGAAGCTGACCATCAGGATAACATTGAGGATAATTCTGAAGATAGCTCTCTAATACCCAGTATTGGTCGAGACAACGCCATTAGCTGTCTCATTCATTGCTCGAGGTCAGATTATGGTGCCATGTCAGCTTTGAACAGGAGCTTCCGCTCATTAATCAGGAGCGGGGATATCTACAAATTAAGACGTATGAATGGTGTCGTTGAGCACTGGGTTTATTTTTCCTGCAACCTGCAGGAATGGGAAGCTTTTGACCCCAACCGTCGTCGTTGGATGCGTCTTCCAACGATGAATTCTAATGAATGCTTCTGGTTTTCCGACAAGGAATCGTTGGCTGTTGGAACGGAGCTCCTCGTGTTCGGAAAGGAGCTAATGGCACAGGTCATTTATCGATACAGTCTGCTGACCAATTCATGGTCATCTGGAATGCAGATGAAAGAGCCACGGTGTTTGTTCGGTTCTTCCAGCAAAGGGGAGATAGCTATTCTTGCTGGCGGTTGTGACTCACATGGTAAGATCCTTAGCTCAGCCGAGCTTTATAACTCCGAGACTGGAACTTGGACACTATTGCCAAATATGAACAAGGCCAGGAAGATGTGTTCCGGGGTTCTCATGGATGGAAAGTTTTACGTGGTTGGAGGCGTTGGAGGACCTGATTCGAAGCTTTTGACATGTGGGGAGGAATATGATTTCGAGAAAAGAACATGGACGGAAATACCAAACATGTCCCCTGTGCGTATGGCTGCAGTTAGGGAAAATGGCTTGCCCGCCACAGCTGAAGCACCTCCTTTGCTTGCAGTTGTAAATAACCAATTGTATGCAGCCGATTATGCTGACATGGAAGTTAGGGGGTATGACAAGGACAAAAAGGCGTGGGTTACTGAAGGAAGATTGCCTGAGCGAGCAAATTCTATGAACGGTTGGGGCTTGGCTTTTCGAGCATGTGGCGACAGGCTTCTTGTAATCGGAGGTCCTAGGACTGTGGGTCAAGGATTTATTGAAGTGAACTCGTGGGTCCCCAGCGAAGGACCGCCACAGTGGAATATGCTCGGCCGAAAGTCATCTGGTAGCTTTGTCTATAATTGTGCGGTGATGGGTTGCTAA
IbTAG2 ATGGCCGGGGGAGGAGGGAGTTCAAGATCAGGAGGAGGAGAAGACAGTGGTGATTCAGGGTGTGGTTTGAATGCTTTTGCAAACAGAGCAGAGGGACCAGAATCCTCCATTAGCACAAAGGCTTCAGACCAACAATCCTTTGACTACCAAAAACAACCTCAATTTCTACAAGCAGAGATGGCAACAAGTGACTCCCCTGCCCCTGGGGGAAATGCTGATCAACCACCTCCTAAACACCCCACTCAAGATAAGAGAAAGGGTGCTGCTTCATCCTCTGATAGAAATCTTGATGCTAAGACACTGAGGCGTTTAGCTCAGAATAGAGAAGCTGCAAGAAAAAGCAGGCTAAGGAAAAAGGCCTATGTACAGCAACTAGAAACCAGTAGGATACGGCTTGCTCAGCTAGAACAGGAGCTTCAAAGAGCACGGTCCCAGGGAATTCTTGTGGGAGGTGGTGCGGCGGCTAGTGCCGGCATCAGCTCTGGGGCTGCCATTTTTGACATGGAATATGCAAGATGGTTAGAGGATGGTCACAGGCACATGTCTGAACTTAGAACAGCACTGCAGGCCCATTTATCAGATGATGATCTGAGGGTGATTATTGATTGCTACATTGCCCACTACGACGAGATTTTCCGGCTCAAAGGGGTGGCGGCAAAGACTGATGTTTTCCATCTCATCACCGGAATGTGGATGACTCAGGCGGAGCGCTGTTTTCTTTGGATGGGTGGTTTTAGGCCATCCGAGCTAATCAAGATGCTGTGTGGGCAGTTAGACCCCTTGACAGAACAGCAAGTTATGGGGATATACAGCCTTCAACATTCTACCCAGCAAGCAGAGGAGGCTCTTACCCAGGGATTGGATCAACTGCAACACTCTTTAGTTGAGACCATTGCTAGTGGCTCACTCAATGATGGCATGCATCATATGGCCATTGCATTGGGCAAGCTCTCAAATCTTGAAGGCTTCATTCGCCAGGCTGATAATTTAAGACAGCAAACCCTTCATCAGTTGTGCCGGATACTGACGGTTAGACAAGCGGTTAGGTGTTTCTTGGTGATTGGAGAGTACTATGGCCGGCTTCGGGCTCTCAGCTCCCTTTGGATATCTCGTCCACCAGAGACAACGTTGGTGATTGACAATGGCGCTTGCCAAGCAACTACGGAGCTACAAATGGTGCAAAACTCTCACAACCAATTCGCAAACTTCTGA
IbERF2 ATGTGCGGTGGTGCTATAATCTCCGATATCAAACCGCCGGTGCGGACGTCGCGCCGGCTGACCTCTGAGTTGCTGTGGGGAAGCGCTGATAGGAGCAGCGCAGGTGGGAAGAAGAAGAAGAATTTTGCTGGAAGCTACTATTCCGGAGCCCTGCGATCCGCGGCTTTTGACCTCGATAATGAATTCGAGGCTGATTTTCAGGACTTCAATGACTATGATGATGATGAGGTCGTAGATGTGGACGTCAAACCCTTCGTTTTCGGACTCTCTTGCGGTTTCAGCTCTGATTCTAAGGACTTTGACCAATCTATAAAGAGGAAGAGGAAAAATCAGTACAGGGGGATCGGGCAGCGTCCTTGGGGCAAATGGGCGGCTGAAATCCGTGATCCGAGTAAAGGTGTTCGAGTCTGGCTAGGAACTTTCGACACTGCTGAAGAAGCGGCGAGAGCCTATGATACTGAGGCTCGGAGGATCAGAGGCAACAAAGCTAAGTTGAATTTCCCTGCTGAAACTCAAGCAAAATCGTCTAGGCGCACCGTTAAGGTGAATCCACGGAAGGTGGTCCCTAAGACGGTTCAACCTGATGCGATTAGCATGGACAATAGCTATTATGGTGGAAGCAACAATTCCTTGGACTGTACTGAGCATGGCTCAAGGACTCCAGAGATATCCTCTGCTCTGTCGGCTGCTGTTGAAGCTGATGAGATTCAGTTCATTCAGGAAAGCAACCCGCAAAAGAAACTGAAGTCAACGGAAAACACCACCGTTGAGAAGCTGTCTGAAGAGCTCTCGGCTTTTGAAGATCAGATGAAGTTCTTTCAGATCCCATATCTGGAGGGGAACTGGGACTCATCAGTTGACACTTACCTTAATGTTGATGGTGGAAACGCCTTAGATCTCTGGTCATTTGATGATGTTCCTTCTTTAATGGGGGGTGTCTTCTAG

表1

IbOPR2启动子中抗逆相关顺式作用元件"

元件
Element		 核心序列
Core sequence (5′-3′)		 功能
Function
MYB TGGTTG 胁迫响应元件 Stress response elements
CGTCA-motif CGTCA 茉莉酸甲酯应答元件 MeJA response elements
MYC CAATTG 胁迫响应元件 Stress response elements
CCAAT-box CCACGG MYBHv1结合位点 MYBHv1 binding site
MYB-binding site CAACAG MYB结合位点 MYB-binding site
W-box TTGACT(C) WRKY结合位点 WRKY binding site

图2

IbOPR2启动子在烟草叶片中的GUS活性分析 A: 清水对照组; B: 试验组。标尺为1 cm。"

图3

Y1HGold [IbOPR2-proMeJA-MYC-pAbAi]菌株的AbA抗性筛选 AbA: 金担子素A。"

图4

酵母单杂交文库筛选阳性克隆的PCR检测 1 -116: 酵母菌落PCR扩增产物。"

图5

IbPHL7、IbZFP16、IbSKIP11、IbTGA2和IbERF2基因的克隆 1 : IbPHL7的扩增产物; 2: IbZFP16的扩增产物; 3: IbSKIP11的扩增产物; 4: IbTGA2的扩增产物; 5: IbERF2的扩增产物。"

图6

酵母单杂交点对点IbPHL7、IbZFP16、IbSKIP11、IbTGA2和IbERF2与IbOPR2启动子互作验证 AbA: 金担子素A。"

图7

IbPHL7、IbZFP16和IbSKIP11激活IbOPR2启动子的双荧光素酶报告基因分析 标尺为1 cm。"

图8

IbPHL7、IbZFP16和IbSKIP11在盐胁迫处理下不同时间的表达分析 根据Duncan检验, 不同小写字母表示在0.05水平差异显著。"

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