香石竹DcERF-1转录因子对切花衰老的负调控作用

doi:10.16420/j.issn.0513-353x.2021-0478

园艺学报 ›› 2022, Vol. 49 ›› Issue (6): 1313-1326.doi: 10.16420/j.issn.0513-353x.2021-0478

香石竹DcERF-1转录因子对切花衰老的负调控作用

王妍¹^,², 孙政¹^,², 冯珊¹^,², 袁心怡¹^,², 仲林林¹^,², 曾云流¹^,², 傅小鹏¹^,⁴, 程运江¹^,²^,³, 包满珠¹^,⁴, 张帆¹^,²^,³^,⁴^,^*()

¹华中农业大学园艺林学学院,园艺植物生物学教育部重点实验室,武汉 430070
²国家柑橘保鲜技术研发专业中心,武汉 430070
³湖北洪山实验室,武汉 430070
⁴华中农业大学园艺林学学院,农业农村部华中都市农业重点实验室,武汉 430070

收稿日期:2021-12-31 修回日期:2022-04-22 出版日期:2022-06-25 发布日期:2022-07-05
通讯作者: 张帆 E-mail:zhangfan@mail.hzau.edu.cn
基金资助:
中央高校基本科研业务费项目(2662019PY049);国家千人计划青年项目;华中农业大学高层次人才启动经费项目

The Negative Regulation of DcERF-1 on Senescence of Cut Carnation

WANG Yan¹^,², SUN Zheng¹^,², FENG Shan¹^,², YUAN Xinyi¹^,², ZHONG Linlin¹^,², ZENG Yunliu¹^,², FU Xiaopeng¹^,⁴, CHENG Yunjiang¹^,²^,³, Bao Manzhu¹^,⁴, ZHANG Fan¹^,²^,³^,⁴^,^*()

¹Key Laboratory of Horticultural Plant Biology,Ministry of Education,College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan 430070,China
²National R & D Center For Citrus Preservation,Wuhan 430070,China
³Hubei Hongshan Laboratory,Wuhan 430070,China
⁴Key Laboratory of Huazhong Urban Agriculture,Ministry of Agriculture and Rural Affairs,College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan 430070,China

Received:2021-12-31 Revised:2022-04-22 Online:2022-06-25 Published:2022-07-05
Contact: ZHANG Fan E-mail:zhangfan@mail.hzau.edu.cn

摘要/Abstract

摘要：

以香石竹（Dianthus caryophyllus L.）为材料,克隆得到1个ERF转录因子基因,命名为DcERF-1。氨基酸序列比对及系统进化树分析发现,DcERF-1与拟南芥AtERF-1的同源性最高,属于Ⅸ（B3）亚组。实时荧光定量PCR显示,DcERF-1表达量在花中最高,且在花瓣自然衰老和乙烯诱导的衰老过程中都呈现先上升后下降的趋势。DcERF-1定位于细胞核中。DcERF-1在香石竹花瓣中瞬时过量表达后,花瓣褪色速度明显延缓,离子渗透率明显降低; DcERF-1被瞬时沉默后,花瓣褪色速度显著加快,离子渗透率显著升高,衰老标志基因DcSAG12表达量显著上调。酵母单杂交试验证明乙烯信号转导途径核心转录因子DcEIN3能直接结合DcERF-1的启动子。双荧光素酶瞬时表达试验证明DcERF-1能抑制乙烯生物合成途径中ACC氧化酶基因DcACO4的表达活性。综合结果表明DcERF-1负调控香石竹切花衰老。

关键词: 香石竹, 切花衰老, 乙烯, 采后, ERF转录因子, 转录调控

Abstract:

In order to explore the mechanism of ethylene regulated flower senescence in cut carnation,we screened a transcription factor named DcERF-1 from the expression profile of carnation. Analysis of deduced amino acid sequence and phylogenetic tree found that DcERF-1 showed high homology with Arabidopsis thaliana AtERF-1,and belongs to Ⅸ（B3）sub-group. Real-time quantitative PCR showed that DcERF-1 was highly expressed in flower,and the expression trend of DcERF-1 was firstly increased and then decreased during both the natural flower senescence and ethylene induced flower senescence progress. The subcellular localization assay showed that DcERF-1 was localized in the nucleus. Transient overexpression of DcERF-1 in the petals resulted that the petal fading rate was significantly delayed and the ion leakage rate was significantly reduced. After transient silencing of DcERF-1,the petal fading rate was significantly accelerated,the ion leakage rate was significantly increased,and the expression of senescence marker gene DcSAG12 was significantly up-regulated. In addition,yeast one hybrid assay showed that DcEIN3 could directly bind the promoter of DcERF-1,and dual luciferase assay showed that DcERF-1 could inhibit the expression of DcACO4. The comprehensive results indicated that DcERF-1 negatively regulates the flower senescence in cut carnation.

Key words: carnation, cut flower senescence, ethylene, postharvest, ERF transcription factor, transcriptional regulation

中图分类号:

S681.5

王妍, 孙政, 冯珊, 袁心怡, 仲林林, 曾云流, 傅小鹏, 程运江, 包满珠, 张帆. 香石竹DcERF-1转录因子对切花衰老的负调控作用[J]. 园艺学报, 2022, 49(6): 1313-1326.

WANG Yan, SUN Zheng, FENG Shan, YUAN Xinyi, ZHONG Linlin, ZENG Yunliu, FU Xiaopeng, CHENG Yunjiang, Bao Manzhu, ZHANG Fan. The Negative Regulation of DcERF-1 on Senescence of Cut Carnation[J]. Acta Horticulturae Sinica, 2022, 49(6): 1313-1326.

图/表 12

表1 引物序列

Table 1 Primer sequences

用途 Usage	名称 Name	引物序列（5′-3′） Primer sequence
基因沉默 Gene silencing	TRV2-DcERF-1	F：cgtgagctcggtaccggatccAGAGGATGCAGCTTTGGCC
基因沉默 Gene silencing		R：gtgagtaaggttaccgaattcCTAATTAACTAACAATTGATCACCAAGTTG
基因过表达 Gene overexpression	p1300-DcERF-1	F：caaatcgactctagaaagcttATGAGTAATTTTGATTCTGATTATACAGTTCT
基因过表达 Gene overexpression		R：gcccttgctcaccatggtaccATTAACTAACAATTGATCACCAAGTTGC
实时荧光定量 PCR Real-time quantitative PCR	DcERF-1-qPCR	F：CAACGTCCTCCTCAGAAACCAG
		R：TCACACCAACTTGACAACCCAC
	DcSAG12-qPCR	F：TCAGAATGACGAAGACGCCC
		R：AACCGCACTCGCCCTTAAAC
	DcACO4-qPCR	F：ACCATGCTTGCGAGAATTGG
		R：AGCGGTTTCCCAGTCAGTTG
	DcACS3-qPCR	F：TTTGATGGGTGGAAAGCTTATG
		R：AAATGGATGCACTTGGGTTCTT
	DcACS9-qPCR	F：GTCTCTTCCTCGGCTCCGTC
		R：ACAAATGGGCCTGACTCGAC
	DcACO1-qPCR	F：GGGAAGTGATGCCGTGATTT
		R：CAAGCAGTTGGAATGGGACC
	DcACS1-qPCR	F：AAGGAAACGCGGCATTGTT
		R：CGAAGCTCCATTTGCCCTC
酵母单杂交 Yeast one hybrid assay	AD-DcEIN3	F：gccatggaggccagtgaattcATGGTGATGTTGTTTGAGGAAATG
		R：atgcccacccgggtggaattcTTATCTGTACCAAGCCGATGATGT
	DcP-pAbAi	F：cttgaattcgagctcggtaccTTTCTACCACAAAATAATGAATTAAATATTC
		R：atacagagcacatgcctcgagTCTGGGTATTGATTACTTAAGCTAAAAA
双荧光素酶试验 Dual luciferase assay	DcERF-1-62-SK	F：caggaattcgatatcaagcttATGAGTAATTTTGATTCTGATTATACAGTTCT
		R：tcagcgtaccgaattggtaccCTAATTAACTAACAATTGATCACCAAGTTG
	DcACS9-0800	F：gtcgacggtatcgataagcttAACACAAATTCTTATTTAAGACGGGTA
		R：cgctctagaactagtggatccAGTTTCTAGTTGAGGTGATAATGATTAGG
	DcACS3-0800	F：gtcgacggtatcgataagcttTTTCTTATCTAATATTCAGTCAATTATTCAATT
		R：cgctctagaactagtggatccAAAAACCACCCCAAGAAAAAAA
	DcACO4-0800	F：gtcgacggtatcgataagcttTCCCAAAGTCCAGCATTTGTTG
		R：cgctctagaactagtggatccTTTTCTTCCTACAAATTAAAATATATTTTACAA

图1 香石竹DcERF-1 基因ORF序列PCR扩增产物

Fig. 1 PCR amplification of ORF sequence of DcERF-1 gene in carnation

图2 香石竹DcERF-1与其他植物ERF蛋白氨基酸比对 Ph：矮牵牛;Ma：香蕉;Md：苹果;At：拟南芥。

Fig. 2 Alignment of the predicted ERF proteins between carnation and other plant species Ph：Petunia hybrida;Ma：Musa acuminata：Md：Malus × domestica;At：Arabidopsis thaliana.

图3 香石竹DcERF-Ⅸ亚组蛋白与拟南芥AtERF-Ⅸ亚组蛋白的进化关系 ●○ 同源蛋白。

Fig. 3 The phylogenetic tree of carnation DcERF-Ⅸ proteins and Arabidopsis thaliana AtERF-Ⅸ proteins ●○ Homologous proteins.

图4 DcERF-1在香石竹不同器官（A）、不同开放阶段（B）和乙烯处理后不同时间的表达量（C）

Fig. 4 The expression level of DcERF-1 in different organs（A）,different flower opening stages（B）and different time after ethylene treatment（C） *α = 0.05;** α = 0.01;*** α = 0.001.

图5 香石竹DcERF-1定位于细胞核中

Fig. 5 DcERF-1 is located in nucleus

图6 DcERF-1瞬时过表达延缓香石竹切花衰老 A：褪色表型;B：表达量;C：离子渗透率。

Fig. 6 Overexpression of DcERF-1 delayed petal senescence A：The color-fading phenotype;B：The expression level;C：The ion leakage rate. ** α = 0.01.

图7 DcERF-1沉默加速花瓣衰老 A：褪色表型;B：表达量;C：离子渗透率。

Fig. 7 Silencing of DcERF-1 promotes petal senescence A：The color-fading phenotype;B：The expression level;C：The ion leakage rate. ** α = 0.01;*** α = 0.001.

图8 DcERF-1启动子序列（A）和酵母单杂交实验（B）

Fig. 8 The promoter sequence of DcERF-1（A）and yeast one hybrid assay（B）

图9 乙烯合成基因在乙烯处理后的表达量

Fig. 9 The expression profile of ethylene synthesis genes after ethylene treatment

图10 DcERF-1抑制DcACO4基因的启动子活性

Fig. 10 DcERF-1 inhibits the promoter activity of DcACO4 * α = 0.05.

图11 香石竹DcERF-1调控切花衰老的预想模型

Fig. 11 The proposed model of DcERF-1 in regulating flower senescence in cut carnation

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香石竹DcERF-1转录因子对切花衰老的负调控作用

The Negative Regulation of DcERF-1 on Senescence of Cut Carnation

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香石竹DcERF-1转录因子对切花衰老的负调控作用

The Negative Regulation of DcERF-1 on Senescence of Cut Carnation

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相关文章 15

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