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作物学报 ›› 2024, Vol. 50 ›› Issue (1): 126-137.doi: 10.3724/SP.J.1006.2024.34045

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花AP2/ERF转录因子GhTINY2负调控植株抗盐性的功能分析

肖胜华1,2,*(), 陆妍1(), 李安子1, 覃耀斌1, 廖铭静1, 闭兆福1, 卓柑锋1, 朱永红2, 朱龙付2,*()   

  1. 1广西大学农学院 / 亚热带农业生物资源保护与利用国家重点实验室, 广西南宁 530000
    2华中农业大学 / 作物遗传改良国家重点实验室, 湖北武汉 430000
  • 收稿日期:2023-03-06 接受日期:2023-06-29 出版日期:2024-01-12 网络出版日期:2023-07-21
  • 通讯作者: *肖胜华, E-mail: shxiao@gxu.edu.cn; 朱龙付, E-mail: lfzhu@mail.hzau.edu.cn
  • 作者简介:肖胜华, E-mail: shxiao@gxu.edu.cn; 陆妍, E-mail: 714675568@qq.com

    **同等贡献

  • 基金资助:
    广西大学高层次人才科研启动基金项目(A3310051044);广西大学农学院科研发展金项目(EE101711)

Function analysis of an AP2/ERF transcription factor GhTINY2 in cotton negatively regulating salt tolerance

XIAO Sheng-Hua1,2,*(), LU Yan1(), LI An-Zi1, QIN Yao-Bin1, LIAO Ming-Jing1, BI Zhao-Fu1, ZHUO Gan-Feng1, ZHU Yong-Hong2, ZHU Long-Fu2,*()   

  1. 1State Key Laboratory of Conservation and Utilization of Agro-Biological Resources in Subtropical Region / College of Agriculture, Guangxi University, Nanning 530000, Guangxi, China
    2State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430000, Hubei, China
  • Received:2023-03-06 Accepted:2023-06-29 Published:2024-01-12 Published online:2023-07-21
  • Contact: *E-mail: shxiao@gxu.edu.cn; E-mail: lfzhu@mail.hzau.edu.cn
  • About author:**Contributed equally to this study
  • Supported by:
    Initial Scientific Research Fund of High-level Personnel in Guangxi University(A3310051044);Guangxi University Agricultural College Research Development Fund(EE101711)

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摘要：

棉花属于相对耐盐作物, 但高盐胁迫同样会造成棉花产量和纤维品质的大幅下降。深入挖掘抗盐基因并解析棉花响应盐胁迫的分子机理, 对加快棉花抗盐遗传改良育种进程具有重要意义。本研究从棉花响应盐胁迫的转录组数据中鉴定到一个受盐诱导极显著下调表达的AP2/ERF转录因子GhTINY2, 并分析了GhTINY2超表达拟南芥的抗盐表型和各生理指标。结果显示, 在盐胁迫下, GhTINY2超表达植株的种子萌发率显著下降; 脯氨酸、可溶性糖、叶绿素含量等均显著减少; 多个盐胁迫响应基因显著下调表达; 因而表现出更为严重的叶片萎蔫枯黄表型。通过分析GhTINY2超表达拟南芥中的RNA-seq数据, 发现差异表达基因(DEGs)富集到叶绿素代谢、刺激响应等生物过程中, 且DEGs均呈下调表达趋势。此外, 在棉花中通过病毒诱导的基因沉默(VIGS)试验沉默GhTINY2后, TRV:GhTINY2植株在盐胁迫下叶绿素和脯氨酸含量显著增加, 从而增强了棉花的抗盐性。综上, GhTINY2是棉花中一个负调控盐胁迫抗性的重要基因, 未来将有望通过现代基因工程技术利用GhTINY2创制耐盐棉花材料。

关键词: 棉花, GhTINY2, 盐胁迫, 转录因子, 转基因

Abstract:

Cotton is a relatively salt-tolerant crop, but high salt stress leads to a significant decline in cotton yield and fiber quality. Mining the genes involved in salt-tolerance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Here, we identified an AP2/ERF transcription factor GhTINY2 in the transcriptome database from cotton treated with salt, and the relative expression level of GhTINY2 was reduced by salt. Subsequently, the salt-resistant phenotype and physiological indicators of the GhTINY2-overexpression Arabidopsis were analyzed. The results revealed that the GhTINY2-overexpression Arabidopsis had a significant decrease in seed germination rate, the content of proline, soluble sugar, and chlorophyll under salt stress, leading to more severe leaf wilting compared with WT. RNA-seq data from GhTINY2-transgenic Arabidopsis revealed that differentially expressed genes (DEGs) were enriched in a series of biological processes, including chlorophyll metabolism and response to stimulus, and the relative expression level of these DEGs significantly was down-regulated. Moreover, the silence of GhTINY2 in cotton through Virus-induced gene silencing (VIGS) assay showed that TRV:GhTINY2 had a significant increase in chlorophyll and proline content, leading to improved salt tolerance compare with TRV:00. In conclusion, these findings suggest that GhTINY2 was an important gene in cotton that negatively regulated salt stress resistance, and it was expected to create salt-tolerant cotton materials using GhTINY2 gene by modern genetic engineering technology in the future.

Key words: cotton, GhTINY2, salt stress, transcription factor, transgenic

表1

本研究所使用的引物信息表"

引物名称Primer name 引物序列Primer sequence (5°-3°)
GhUB7-F GAAGGCATTCCACCTGACCAAC
GhUB7-R CTTGACCTTCTTCTTCTTGTGCTTG
AtACTIN2-F AAATCACAGCACTTGCACCAAGC
AtACTIN2-R GGCCTTGGAGATCCACATCTGC
AtP5CS2-F ATGATCTTATTTATGTTCTGC
AtP5CS2-R CACTATCTTCCGTCACTAT
AtP5CS1-F ACCAGAAGCACGGTCATTC
AtP5CS1-R CCATCTGAGAATCTTGTG
AtProDH2-F AAGTGTCAGCATCACAAC
AtProDH2-R CACGAAGAAATCATCAC
AtRD20-F GATGGAATCGTCTATCCTTGGG
AtRD20-R ACTGGGACATACCTTCCTTCGG
AtRD22-F CCCATTCCCAACTCTCTCCAT
AtRD22-R GACCTTTTCCGCTGCCAAC
AtRD26-F ATGGGTCGTCATCGTCTTCTTC
AtRD26-R GAAACGCATCGTAACCACCG
TRV:GhTINY2-F GCGTGAGCTCGGTACCGGAGGAGCTGAGCCAGATAGTG
TRV:GhTINY2-R GCCTCCATGGGGATCCCTAGAAATTTTGACCTATCCATGCTA
GhTINY2-F CATCTTCATCGTTGTCGTCCTCA
GhTINY2-R CAGTTTTCTTCGTAATACCAAGGCAT

图1

GhTINY2受NaCl和ABA诱导的表达模式 A: GhTINY2受NaCl诱导的表达热图, 红色表示基因高表达、白色表示基因低表达。B~C: RT-qPCR检测GhTINY2受NaCl (B)和ABA (C)诱导后的表达量。星号代表显著差异(**, P < 0.01, 采用t检验)。"

图2

GhTINY2抑制盐胁迫下的拟南芥种子萌发 A: GhTINY2超表达拟南芥种子在含NaCl和不含NaCl的培养基中萌发2周的表型。B~C: 统计GhTINY2超表达拟南芥种子在不含NaCl (B)和含NaCl (C)培养基中的萌发率。标尺为1 cm, 星号代表显著差异(**, P < 0.01, 采用t检验)。"

图3

GhTINY2负调控拟南芥的盐胁迫抗性 A: GhTINY2超表达拟南芥在含NaCl营养土中的生长表型。B: GhTINY2超表达拟南芥在盐胁迫下的存活率。C: GhTINY2超表达拟南芥离体叶片的失水率。星号代表显著差异(**: P < 0.01, 采用t检验)。"

图4

盐胁迫下GhTINY2超表达拟南芥的各生理指标含量的测定 A~D: GhTINY2超表达拟南芥在盐胁迫下MDA (A)、可溶性糖(B)、脯氨酸(C)和叶绿素(D)含量的测定。星号代表显著差异(**: P < 0.01, 采用t检验)。"

图5

GhTINY2超表达拟南芥的RNA-seq分析 A: GhTINY2超表达拟南芥RNA-seq数据中的差异表达基因火山图。B~C: GhTINY2超表达拟南芥差异表达基因的KEGG富集 (B)和GO分类(C)。"

图6

GhTINY2超表达拟南芥中差异表达基因热图 热图中的数字表示GhTINY2-OE相较于WT的差异表达基因下调倍数。"

图7

GhTINY2超表达拟南芥中盐胁迫响应基因的表达分析 A~B: 在正常条件和盐胁迫条件下, WT和GhTINY2超表达拟南芥中盐胁迫标志基因(A)和脯氨酸合成/降解基因(B)的表达水平。星号代表显著差异(*: P < 0.05; **: P < 0.01, 采用t检验)。"

图8

在棉花中沉默GhTINY2可增强盐胁迫抗性 A: RT-qPCR检测GhTINY2的沉默效果。TRV:00表示空载体对照棉花植株, TRV:GhTINY2表示GhTINY2沉默后的棉花植株。B: TRV:00和TRV:GhTINY2棉花植株的抗盐表型。C~D: 在盐胁迫下TRV:00和TRV:GhTINY2植株叶片中叶绿素(C)和脯氨酸(D)含量。星号代表显著差异(**: P < 0.01, 采用t检验)。"

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