胡萝卜番茄红素ε-环化酶基因DcLCYE启动子活性分析及互作因子筛选

doi:10.16420/j.issn.0513-353x.2024-0750

园艺学报 ›› 2025, Vol. 52 ›› Issue (9): 2317-2328.doi: 10.16420/j.issn.0513-353x.2024-0750

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

胡萝卜番茄红素ε-环化酶基因DcLCYE启动子活性分析及互作因子筛选

王雅慧¹, 张雨晴¹, 张榕蓉¹, 刘佩卓¹, 梁毅², 周建华³, 熊爱生¹^,^*()

¹ 南京农业大学园艺学院，作物遗传与种质创新利用全国重点实验室，农业农村部华东地区园艺作物生物学与种质创制重点实验室，南京 210095
² 北京市农林科学院蔬菜研究所，北京 100097
³ 郑州市农业科技研究院，郑州 450040

收稿日期:2025-04-17 修回日期:2025-07-21 出版日期:2025-09-25 发布日期:2025-09-24
通讯作者:
^* E-mail：xiongaisheng@njau.edu.cn
基金资助:
国家自然科学基金青年科学基金项目(32402551); 江苏省自然科学基金青年基金项目(BK20241562); 国家资助博士后研究人员计划项目(GZC20240721); 江苏省卓越博士后计划项目(2024ZB102); 江苏省高校优势学科项目(PAPD)

Analysis of Promoter Activity and Screening of Interaction Factors of Carrot Lycopene ε-Cyclase Gene DcLCYE

WANG Yahui¹, ZHANG Yuqing¹, ZHANG Rongrong¹, LIU Peizhuo¹, LIANG Yi², ZHOU Jianhua³, XIONG Aisheng¹^,^*()

¹ College of Horticulture， Nanjing Agricultural University/State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization/Ministry of Agriculture and Rural Affairs Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China， Nanjing 210095， China
² Beijing Vegetable Research Center of Beijing Academy of Agriculture and Forestry Sciences， Beijing 100097， China
³ Zhengzhou Agricultural Science and Technology Research Institute， Zhengzhou 450050， China

Received:2025-04-17 Revised:2025-07-21 Published:2025-09-25 Online:2025-09-24

摘要/Abstract

摘要： 分别从胡萝卜‘黑田五寸’‘红玉八号’‘本红金时’‘玫瑰红’‘大禹美人指’和‘贝塔’中克隆得到长度为970 bp的DcLCYE基因启动子序列，并对其进行生物信息分析。结果显示，胡萝卜DcLCYE基因启动子区域含有多种光响应元件、激素响应元件以及转录因子结合位点等。利用不同长度DcLCYE基因启动子缺失片段驱动GUS报告基因表达，在胡萝卜中进行稳定表达检测启动子活性，结果表明起始密码子上游610 bp长的启动子区域具有较强的活性。对不同胡萝卜品种DcLCYE基因启动子进行序列比较，发现表型相近的品种DcLCYE基因启动子序列相似性更高，启动子差异可能会影响DcLCYE在不同胡萝卜品种中的转录。构建胡萝卜cDNA文库，利用DcLCYE启动子诱饵载体，采用酵母单杂交系统筛选与DcLCYE基因启动子互作的上游调控因子，阳性克隆包括数个抗氧化酶蛋白、热激蛋白、锌指蛋白和水解酶蛋白等，还筛选到可能在胡萝卜类胡萝卜素积累调控中发挥作用的MYB、bHLH以及bZIP等转录因子。

关键词: 胡萝卜, 番茄红素, 启动子, 酵母单杂交, 转录因子

Abstract:

The 970 bp promoter sequence of DcLCYE was cloned and bioinformatics analysis was carried out from carrot‘Kurodagosun’‘Hongyu 8’‘Benhong Jinshi’‘Meiguihong’‘Dayu Meirenzhi’and‘Beita’，respectively. The results showed that the promoter region of carrot DcLCYE contained various light response elements，hormone response elements and transcription factor binding sites. GUS reporter expression was driven by promoter deletion fragments of DcLCYE with different lengths，and promoter activity was detected by stable expression in carrot. The results showed that the promoter region of 610 bp upstream of the initiation codon had stronger promoter activity. By comparing the sequences of DcLCYE promoters of different carrot varieties，it was found that the sequences of DcLCYE promoters of carrot varieties with similar phenotypes were more consistent. The differences of promoters may affect the transcription of DcLCYE in different carrot varieties. A carrot cDNA library was constructed，and the upstream regulatory factors interacting with DcLCYE promoter were screened by yeast one-hybrid system. Positive clones included several antioxidant proteins，heat shock proteins，zinc finger proteins，hydrolase proteins，etc. Transcription factors such as MYB，bHLH and bZIP，which may play roles in the regulation of carrot carotenoid accumulation were also screened.

Key words: carrot, lycopene, promoter, yeast one-hybrid, transcription factor

王雅慧, 张雨晴, 张榕蓉, 刘佩卓, 梁毅, 周建华, 熊爱生. 胡萝卜番茄红素ε-环化酶基因DcLCYE启动子活性分析及互作因子筛选[J]. 园艺学报, 2025, 52(9): 2317-2328.

WANG Yahui, ZHANG Yuqing, ZHANG Rongrong, LIU Peizhuo, LIANG Yi, ZHOU Jianhua, XIONG Aisheng. Analysis of Promoter Activity and Screening of Interaction Factors of Carrot Lycopene ε-Cyclase Gene DcLCYE[J]. Acta Horticulturae Sinica, 2025, 52(9): 2317-2328.

图/表 9

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顺式作用元件 cis-Element	核心序列 Core sequence	位置（ATG前）/bp Position from ATG	方向 Direction	功能描述 Function description
TATA-box	TATA	333；343；459；562；690；716；766	+；+；+；+；+；+；+	转录起始位点前30 bp核心启动子元件 Core promoter element around -30 bp of transcription start
CAAT-box	CAAT	61；115；153；303；398；616；680；720； 730；735；758；772；782；878；941	+；-；+；+；-；-；+；+； -；-；-；+；-；-；+	启动子和增强区域顺式作用元件 Common cis-acting element in promoter and enhancer regions
ABRE	ACGTG	191；220；255	+；+；-	脱落酸响应顺式作用元件 cis-Acting element involved in the abscisic acid responsiveness
ABRE	CACGTG	139	+	脱落酸响应顺式作用元件 cis-Acting element involved in the abscisic acid responsiveness
AE-box	AGAAACTT	74	+	光响应元件Part of a module for light response
Box Ⅱ	CCACGTGGC	140	+	光响应元件Part of a light responsive element
CCAAT-box	CAACGG	924	-	MYBHv1结合位点MYBHv1 binding site
CGTCA-motif	CGTCA	935	+	茉莉酸甲酯顺式作用元件cis-Acting regulatory element involved in theMeJA-responsiveness
G-box	CACGTC	139；255	-；+	光响应顺式作用元件 cis-Acting regulatory element involved in light responsiveness
G-box	TACGTG	192	+	光响应顺式作用元件 cis-Acting regulatory element involved in light responsiveness
G-box	CACGTT	221	-	光响应顺式作用元件 cis-Acting regulatory element involved in light responsiveness
GATA-motif	GATAGGG	107	-	光响应元件 Part of a light responsive element
GCN4_motif	TGAGTCA	876	-	参与胚乳表达的顺式作用元件 cis-Regulatory element involved in endosperm expression
MBS	CAACTG	124	-	参与干旱诱导的MYB结合位点 MYB binding site involved in drought-inducibility
MRE	AACCTAA	531；576	-；-	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness
MYC	CATTTG	783	+	MYC识别元件 MYC recognition element
MYB recognition site	CCGTTG	924	+	MYB结合位点 MYB recognition site
TC-rich repeats	GTTTTCTTAC	844	-	防御和逆境响应相关顺式作用元件 cis-Acting element involved in defense and stress responsiveness
TCT-motif	TCTTAC	523	-	光响应元件Part of a light responsive element
TGA-element	AACGAC	837；913	+；+	生长素响应元件Auxin-responsive element

顺式作用元件 cis-Element	核心序列 Core sequence	位置（ATG前）/bp Position from ATG	方向 Direction	功能描述 Function description
TATA-box	TATA	333；343；459；562；690；716；766	+；+；+；+；+；+；+	转录起始位点前30 bp核心启动子元件 Core promoter element around -30 bp of transcription start
CAAT-box	CAAT	61；115；153；303；398；616；680；720； 730；735；758；772；782；878；941	+；-；+；+；-；-；+；+； -；-；-；+；-；-；+	启动子和增强区域顺式作用元件 Common cis-acting element in promoter and enhancer regions
ABRE	ACGTG	191；220；255	+；+；-	脱落酸响应顺式作用元件 cis-Acting element involved in the abscisic acid responsiveness
ABRE	CACGTG	139	+	脱落酸响应顺式作用元件 cis-Acting element involved in the abscisic acid responsiveness
AE-box	AGAAACTT	74	+	光响应元件Part of a module for light response
Box Ⅱ	CCACGTGGC	140	+	光响应元件Part of a light responsive element
CCAAT-box	CAACGG	924	-	MYBHv1结合位点MYBHv1 binding site
CGTCA-motif	CGTCA	935	+	茉莉酸甲酯顺式作用元件cis-Acting regulatory element involved in theMeJA-responsiveness
G-box	CACGTC	139；255	-；+	光响应顺式作用元件 cis-Acting regulatory element involved in light responsiveness
G-box	TACGTG	192	+	光响应顺式作用元件 cis-Acting regulatory element involved in light responsiveness
G-box	CACGTT	221	-	光响应顺式作用元件 cis-Acting regulatory element involved in light responsiveness
GATA-motif	GATAGGG	107	-	光响应元件 Part of a light responsive element
GCN4_motif	TGAGTCA	876	-	参与胚乳表达的顺式作用元件 cis-Regulatory element involved in endosperm expression
MBS	CAACTG	124	-	参与干旱诱导的MYB结合位点 MYB binding site involved in drought-inducibility
MRE	AACCTAA	531；576	-；-	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness
MYC	CATTTG	783	+	MYC识别元件 MYC recognition element
MYB recognition site	CCGTTG	924	+	MYB结合位点 MYB recognition site
TC-rich repeats	GTTTTCTTAC	844	-	防御和逆境响应相关顺式作用元件 cis-Acting element involved in defense and stress responsiveness
TCT-motif	TCTTAC	523	-	光响应元件Part of a light responsive element
TGA-element	AACGAC	837；913	+；+	生长素响应元件Auxin-responsive element

序列相似性/% Sequence identity	红玉八号Hongyu 8	本红金时Benhong Jinshi	玫瑰红Meiguihong	大禹美人指Dayu Meirenzhi	贝塔Beita
红玉八号Hongyu 8	-
本红金时Benhong Jinshi	87.05	-
玫瑰红Meiguihong	77.47	83.05	-
大禹美人指Dayu Meirenzhi	75.47	80.63	94.42	-
贝塔Beita	75.89	82.32	78.00	77.79	-

序列相似性/% Sequence identity	红玉八号Hongyu 8	本红金时Benhong Jinshi	玫瑰红Meiguihong	大禹美人指Dayu Meirenzhi	贝塔Beita
红玉八号Hongyu 8	-
本红金时Benhong Jinshi	87.05	-
玫瑰红Meiguihong	77.47	83.05	-
大禹美人指Dayu Meirenzhi	75.47	80.63	94.42	-
贝塔Beita	75.89	82.32	78.00	77.79	-

序号 Number	基因登录号 GenBank No.	基因注释 Gene annotation
2	XM_017374601.1	过氧化物酶42 Peroxidase 42（LOC108204913）
3	XM_017364627.1	bHLH68转录因子 Transcription factor bHLH68-like（LOC108197116）
5	XM_017376508.1	DIVARICATA转录因子 Transcription factor DIVARICATA-like（LOC108206263）
7	XM_017375594.1	金属硫蛋白2型 Metallothionein-like protein type 2（LOC108205616）
8	XM_017373362.1	外被体蛋白亚基 Coatomer subunit gamma-2-like（LOC108204071）
9	XM_017370825.1	热休克蛋白70 kD Heat shock 70 kD protein（LOC108202443）
10/49	XM_017382324.1	DIVARICATA转录因子 Transcription factor DIVARICATA（LOC108210874）
17	XM_017382324.1	DIVARICATA转录因子 Transcription factor DIVARICATA（LOC108210874）
18	XM_017387693.1	抗烟草花叶病毒蛋白 TMV resistance protein N-like（LOC108215271）
19	XM_017374468.1	剪切和多聚腺苷酸化特异性因子 30-kD Cleavage and polyadenylation specificity factor 30-like（LOC108204833）
21	XM_017387865.1	木葡聚糖内转葡聚糖酶/水解酶 Xyloglucan endotransglucosylase/hydrolase protein 9-like（LOC108215374）
24	XM_017380200.1	多聚泛蛋白 Polyubiquitin-like（LOC108209349）
26/29	XM_017391471.1	过氧化氢酶 Catalase-like（LOC108218502）
30	XM_017372916.1	乙酰基鸟氨酸脱乙酰基酶 Acetylornithine deacetylase（LOC108192319）
31	XM_017373604.1	液泡加工酶 Vacuolar-processing enzyme-like（LOC108204253）
35	XM_017369114.1	MYB相关蛋白 Myb-related protein Myb4-like（LOC108200851）
37	XM_017403072.1	钠/氢交换因子 Sodium/hydrogen exchanger 6-like（LOC108227751）
38/61	XM_017394896.1	MYB相关蛋白 Myb-related protein 306-like（LOC108220992）
50	XM_017375990.1	MYB59转录因子 Transcription factor MYB59-like（LOC108205875）
54	XM_017402470.1	DNA损伤修复/耐受蛋白 DNA-damage-repair/toleration protein DRT100（LOC108227365）
55/56	XM_017391432.1	2-酮戊二酸脱氢酶 2-Oxoglutarate dehydrogenase（LOC108218476）
59	XM_017380911.1	液泡内在蛋白1-1 Aquaporin TIP1-1（LOC108209795）
60	XM_017388034.1	核糖体蛋白 40S Ribosomal protein S4（LOC108215549）
64	XM_017371878.1	多聚泛素蛋白 Polyubiquitin（LOC108203124）
99	XM_017392716.1	含A20/AN1锌指结构域的胁迫相关蛋白 Zinc finger A20 and AN1 domain-containing stress-associated protein 8-like（LOC108219330）
103	XM_017378127.1	丝裂原活化蛋白激酶 Mitogen-activated protein kinase（LOC108207693）
105	XM_017399617.1	Ras相关蛋白 Ras-related protein Rab11D-like（LOC108224872）
107	XM_017399150.1	Walls Are Thin 1蛋白 WALLS ARE THIN 1-like（LOC108224507）
108	XM_017371475.1	泛醌生物合成酶 Ubiquinone biosynthesis monooxygenase COQ6（LOC108202878）
109	XM_017398063.1	苹果酸脱氢酶 Malate dehydrogenase 1（LOC108223687）
110	XM_017397966.1	水通蛋白PIP2-1 Aquaporin PIP2-1-like（LOC108223626）
126/151	XM_017366601.1	MYB80转录因子 Transcription factor MYB80（LOC108198824）
136	XM_017371867.1	叶绿体移位酶159 Translocase of chloroplast 159（LOC108203115）
153	XM_017386984.1	锌指蛋白 Zinc finger protein CONSTANS-LIKE 1-like（LOC108214788）
155	XM_017392060.1	CBL互作蛋白 CBL-interacting protein kinase 2-like（LOC108218901）
158	XM_017360450.1	ADP核糖基化因子蛋白 ADP-ribosylation factor-like protein 8A（LOC108193681）
176	XM_017388767.1	MYB48转录因子 Transcription factor MYB48（LOC108216091）
183	XM_017371207.1	δ（24）-固醇还原酶 δ（24）-sterol reductase（LOC108202693）
185	XM_017399242.1	β-脲基丙酸酶 β-Ureidopropionase（LOC108224577）
187	XM_017402267.1	2,3-二磷酸甘油酸合成酶 2,3-Bisphosphoglycerate-independent phosphoglycerate mutase（LOC108227225）
188	XM_017368423.1	bZIP转录因子53 bZIP transcription factor 53-like（LOC108200313）
191	XM_017396704.1	HEAT重复内含蛋白 HEAT repeat-containing protein 5B（LOC108222790）

胡萝卜番茄红素ε-环化酶基因DcLCYE启动子活性分析及互作因子筛选

Analysis of Promoter Activity and Screening of Interaction Factors of Carrot Lycopene ε-Cyclase Gene DcLCYE

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胡萝卜番茄红素ε-环化酶基因DcLCYE启动子活性分析及互作因子筛选

Analysis of Promoter Activity and Screening of Interaction Factors of Carrot Lycopene ε-Cyclase Gene DcLCYE

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