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作物学报 ›› 2019, Vol. 45 ›› Issue (3): 469-476.doi: 10.3724/SP.J.1006.2019.84082

• 研究简报 • 上一篇    下一篇

棉花中GhTFL1a和GhTFL1c基因的表达及启动子分析

张晓红1,胡根海1,王寒涛2,王聪聪2,魏恒玲2,付远志1,喻树迅2,*()   

  1. 1河南科技学院生命科技学院 / 现代生物育种协同创新中心, 河南新乡 453003
    2中国农业科学院棉花研究所 / 棉花生物学国家重点实验室, 河南安阳 455000
  • 收稿日期:2018-06-20 接受日期:2018-12-24 出版日期:2019-03-12 网络出版日期:2019-01-04
  • 通讯作者: 喻树迅
  • 基金资助:
    本研究由河南省高等学校重点科研项目(18A210002);棉花生物学国家重点实验室开放课题基金资助(CB2018A08)

Expression and promoter activity of GhTFL1a and GhTFL1c in Upland cotton

Xiao-Hong ZHANG1,Gen-Hai HU1,Han-Tao WANG2,Cong-Cong WANG2,Heng-Ling WEI2,Yuan-Zhi FU1,Shu-Xun YU2,*()   

  1. 1 College of Life Science and Technology, Henan Institute of Science and Technology / Henan Collaborative Innovation Center of Modern Biological Breeding, Xinxiang 453003, Henan, China
    2 Institute of Cotton Research, Chinese Academy of Agricultural Sciences / State Key Laboratory of Cotton Biology, Anyang 455000, Henan, China
  • Received:2018-06-20 Accepted:2018-12-24 Published:2019-03-12 Published online:2019-01-04
  • Contact: Shu-Xun YU
  • Supported by:
    This study was supported by the Key Scientific Research Projects of Colleges and Universities in Henan(18A210002);the State Key Laboratory of Cotton Biology Open Fund(CB2018A08)

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摘要：

从陆地棉中克隆了磷脂酰乙醇胺结合蛋白GhTFL1a和GhTFL1c基因, 并对该基因进行表达分析、启动子预测和启动子活性研究。利用启动子分析软件PlantCARE预测得出, GhTFL1a启动子区域有脱落酸响应元件、干旱诱导的MYB结合位点和顶芽特异表达响应元件等; GhTFL1c启动子区域有乙烯响应元件、干旱诱导的MYB结合位点和水杨酸响应元件。因此, 将pGhTFL1a和pGhTFL1c分别构建到启动子检测载体pBI121-GUS上形成融合表达载体, 通过烟草瞬时转化检测得出这2个基因的启动子都具有活性。实时荧光定量 PCR分析表明, GhTFL1a和GhTFL1c在光周期处理和不同材料的陆地棉(栽培种和半野生种)中表达模式呈相反趋势。GhTFL1a基因受脱落酸(abscisic acid, ABA)、水杨酸(salicylic acid, SA)和盐胁迫诱导, 而GhTFL1c可以响应赤霉素(gibberellin, GA)、SA和ABA胁迫。研究结果初步表明, GhTFL1a和GhTFL1c可能参与了植物逆境胁迫脱落酸和水杨酸响应的调控, 为在棉花中进一步阐明其功能奠定了基础。

关键词: 陆地棉, GhTFL1a, GhTFL1c, 表达分析, 启动子活性

Abstract:

In this study, we cloned the phosphatidylethanolamine-binding protein GhTFL1a and GhTFL1c genes from Upland cotton, and analyzed their expression and promoter activity. The results of promoter structure prediction revealed that GhTFL1a promoter contains abscisic acid (ABA) responsiveness elements, drought-induced MYB binding sites and shoot-specific expression and light responsiveness elements, and the promoter region of GhTFL1c contains ethylene-responsive element, drought-induced MYB binding sites and salicylic acid (SA) responsiveness elements. Thus, we constructed the fusion vector pBI121-GhTFL1a-GUS and pBI121-GhTFL1c-GUS, respectively. Transient transformation of tobacco showed that both promoters had the activity to drive the expression of target gene GUS. Quantitative Real-time PCR result indicated that the expression profile of GhTFL1a and GhTFL1c was opposite during different photoperiod treatments of cultivated and semi-wild cotton. Meanwhile, the expression of GhTFL1a was induced by ABA, SA, and salt (NaCl), while GhTFL1c expression was induced by SA, gibberellin (GA) and ABA. Taken together, the results suggest that GhTFL1a and GhTFL1c might be involved in the regulation of response to abiotic stresses (SA and ABA), which could provide a solid foundation for further function identification.

Key words: upland cotton, GhTFL1a, GhTFL1c, expression analysis, promoter activity

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Purpose
GhTFL1a-up TCCCTGAGCCACTTACCGTTG 荧光定量PCR qRT-PCR
GhTFL1a-down AAGCGTCTCTCATGTCGTTG 荧光定量PCR qRT-PCR
GhTFL1c-up TCATCTGTTGCCACCAAACCT 荧光定量PCR qRT-PCR
GhTFL1c-down TTCCCTTCCAAACGTGGCATC 荧光定量PCR qRT-PCR
GhACTIN-up ATCCTCCGTCTTGACCTTG 荧光定量PCR qRT-PCR
GhACTIN-down TGTCCGTCAGGCAACTCAT 荧光定量PCR qRT-PCR
GhTFL1a-up ATGTCAAGGGTCCCTGAG 基因克隆 Gene cloning
GhTFL1a-down TTATCTTCTTCTTGCAGCAGTTTC 基因克隆 Gene cloning
GhTFL1c-up ATGGGAGAGCCTCTCATTGTT 基因克隆 Gene cloning
GhTFL1c-down TTAGCGTCTCCTTGCAGCAGT 基因克隆 Gene cloning
pGhTFL1a-up AAGGAATATAGAGCACAACA 启动子克隆 Promoter cloning
pGhTFL1a-down GATGAACAAGACGATGTGTAT 启动子克隆 Promoter cloning
pGhTFL1c-up AGTTTAGATTCTTGTGCGAT 启动子克隆 Promoter cloning
pGhTFL1c-down TCTTGATGACAGTGAATGAA 启动子克隆 Promoter cloning

图1

GhTFL1a和GhTFL1c的进化树及氨基酸多重序列比对 A: 进化树分析; B: 氨基酸多重序列比对。*代表氨基酸完全一致, AtBFT、AtTFL1和AtATC序列号分别为NM_125597、U77674和NM_128315, GhTFL1a和GhTFL1c在陆地棉中序列号分别为Gh_A11G0088和Gh_D04G0971。"

图2

GhTFL1a和GhTFL1c在光周期处理试验中的表达模式分析 A和B为GhTFL1a和GhTFL1c基因在栽培种“中棉所36” 中的表达; C和D为GhTFL1a和GhTFL1c基因在半野生种“尖斑棉”中的表达。误差棒为3次重复的标准差。"

图3

GhTFL1a和GhTFL1c在栽培种和半野生种中表达模式分析 横坐标代表取样时期, 分别为长日照子叶、一叶、二叶、三叶、四叶、五叶以及短日照三叶、四叶和五叶。"

表2

陆地棉基因GhTFL1a和GhTFL1c启动子顺式作用元件预测"

基因
Gene		 元件
Element		 序列
Sequence (5′-3′)		 功能
Function
GhTFL1a CE3 GACGCGTGTC ABA和VP1响应元件 ABA and VP1 responsiveness element
MBS TAACTG 干旱诱导的MYB结合位点 MYB binding site involved in drought inducibility
Nodule-site2 CTTAAATTATTTATTT 节结特异因子结合位点 Nodolue specific factor binding site
as-2-box GATAatGATG 顶芽特异表达响应元件 Shoot specific expression element
as-2-box GATAatGATG 顶芽特异表达响应元件 Shoot specific expression element
Circadian CAANNNNATC 昼夜节律控制元件 Circadian rhythms element
GhTFL1c ERE ATTTCAAA 乙烯响应元件 Ethylene responsiveness element
ERE ATTTCAAA 乙烯响应元件 Ethylene responsiveness element
MBS CAACTG 干旱诱导的MYB结合位点 MYB binding site involved in drought inducibility
circadian CAANNNNATC 昼夜节律控制元件 Circadian rhythms element
TCA-element TCAGAAGAGG 水杨酸响应元件 Salicylic acid responsiveness element
TCA-element CAGAAAAGGA 水杨酸响应元件 Salicylic acid responsiveness element

图4

棉花水培苗经过处理后24 h内GhTFL1a基因表达量变化 A: 水杨酸; B: 脱落酸; C: 赤霉素; D: NaCl。误差棒为3次重复的标准差。"

图5

棉花水培苗经过处理后24小时内GhTFL1c基因表达量变化 A: 水杨酸; B: 赤霉素; C: 脱落酸; D: NaCl。误差棒为3次重复的标准差。"

图6

烟草瞬时表达GhTFL1a和GhTFL1c的启动子 A: 阳性对照pBI121; B: pGhTFL1a∷GUS; C: pGhTFL1c∷GUS; D: 阴性对照。"

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