葡萄细胞分裂素响应调节因子VlRRA1的克隆、表达及启动子活性分析

doi:10.16420/j.issn.0513-353x.2022-0516

摘要/Abstract

摘要：

在‘巨峰’葡萄中克隆获得细胞分裂素响应调节因子VlRRA1及其启动子，分析了VlRRA1的表达特征，并对其启动子进行活性分析。结果显示，VlRRA1的cDNA全长为666 bp，编码221个氨基酸;保守结构域预测结果显示该基因仅含有1个磷酸接受结构域（REC），属于A型RR基因;系统进化关系表明VlRRA1与其他物种中的A型RR亲缘关系较近。酵母自激活试验说明VlRRA1不具有转录激活活性，符合A型RR基因的典型特征。qRT-PCR结果表明VlRRA1具有组织表达特异性，主要在茎、叶以及幼果中表达;外源细胞分裂素氯吡苯脲（CPPU）可以促进VlRRA1的表达，而细胞分裂素生物合成抑制剂洛伐他汀（lovastatin，LOV）则抑制VlRRA1的转录。VlRRA1启动子上有多种与非生物胁迫和激素响应相关的元件。GUS组织染色的结果表明，VlRRA1启动子具有活性，可以响应多种激素信号，包括与坐果相关的细胞分裂素、生长素和赤霉素。以上结果表明VlRRA1在细胞分裂素介导的葡萄坐果与幼果发育中具有重要作用。

关键词: 葡萄, 细胞分裂素, 细胞分裂素响应调节因子, VlRRA1, 启动子活性

Abstract:

The cytokinin response regulator VlRRA1 and its promoter were cloned from‘Kyoho’grape. The expression characteristics of VlRRA1 and the activity of VlRRA1 promoter were analyzed. The results showed that the full-length cDNA of VlRRA1 was 666 bp and encoded 221 amino acids. Conservative domain analysis showed that the gene contained only one phosphoacceptor receiver domain（REC），which belongs to type-A RR genes and can only accept phosphate groups;phylogenetic relationships showed that VlRRA1 was closely related to type-A RR in other species. The yeast self-activation assay illustrates that VlRRA1 has no transcriptional activation activity in agreement with other A-type RR genes and does not have a function similar to that of the B-type RR transcription factor. The results of qRT-PCR showed that VlRRA1 had tissue-specific expression and was mainly expressed in stems，leaves and young fruit，slightly lower expression in inflorescence and ripe fruit，suggesting that VlRRA1 may play an important role in the process of fruit set;exogenous cytokinin forchlorfenuron（CPPU）can promote VlRRA1 expression，while the cytokinin biosynthesis inhibitor lovastatin（LOV）inhibits VlRRA1 transcription. Many elements related to abiotic stress and hormone response were found in VlRRA1 promoter. The results of GUS tissue staining showed that VlRRA1 promoter had promoter activity and could respond to a variety of hormone signals，including three plant hormones related to fruit setting，cytokinin，auxin and gibberellin. These results suggest that VlRRA1 plays an important role in grape fruit setting and young fruit development mediated by cytokinin.

Key words: grape, cytokinin, cytokinin response regulator, VlRRA1, promoter activity

李松琦, 李旭飞, 李敏, 刘海楠, 裴茂松, 韦同路, 郭大龙, 余义和. 葡萄细胞分裂素响应调节因子VlRRA1的克隆、表达及启动子活性分析[J]. 园艺学报, 2023, 50(8): 1609-1621.

LI Songqi, LI Xufei, LI Min, LIU Hainan, PEI Maosong, WEI Tonglu, GUO Dalong, YU Yihe. Cloning，Expression and Promoter Activity Analysis of VlRRA1 Gene in Grape[J]. Acta Horticulturae Sinica, 2023, 50(8): 1609-1621.

图/表 10

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用途 Use	引物名称 Name of primer	引物序列（5′-3′） Primer sequence
VlRRA1克隆	VlRRA1-F	atggccatggaggccgaattcATGGAGATAAGCGGTGTTCT
Cloning of VlRRA1 sequence	VlRRA1-R	atgcggccgctgcaggaattcCTAATCACATGTGGAGGTTT
实时荧光定量PCR	qPCR-VlRRA1-F	GACAGATGTTTGGAGGAAGGTG
qRT-PCR	qPCR-VlRRA1-R	TGAAGATATGGACGGTGGTGAT
VlRRA1启动子克隆	ProVlRRA1-F	tgggcccggcgcgccaagcttGGTCATTTCATTGCTTTCCTCTC
Cloning of VlRRA1 promoter sequence	ProVlRRA1-R	ggtggactcctcttagaattcACAGGGTTCCGTGATTCTGTG

用途 Use	引物名称 Name of primer	引物序列（5′-3′） Primer sequence
VlRRA1克隆	VlRRA1-F	atggccatggaggccgaattcATGGAGATAAGCGGTGTTCT
Cloning of VlRRA1 sequence	VlRRA1-R	atgcggccgctgcaggaattcCTAATCACATGTGGAGGTTT
实时荧光定量PCR	qPCR-VlRRA1-F	GACAGATGTTTGGAGGAAGGTG
qRT-PCR	qPCR-VlRRA1-R	TGAAGATATGGACGGTGGTGAT
VlRRA1启动子克隆	ProVlRRA1-F	tgggcccggcgcgccaagcttGGTCATTTCATTGCTTTCCTCTC
Cloning of VlRRA1 promoter sequence	ProVlRRA1-R	ggtggactcctcttagaattcACAGGGTTCCGTGATTCTGTG

顺式作用元件	序列	功能	数量
cis-acting element	Sequence	Function	Number
CAAT-box	CCAAT	启动子和增强子区域常见的顺式作用元件	14
		Common cis-acting element in promoter and enhancer regions
TATA-box	TATAAAT/TATA	转录起始位点周围 - 30左右的核心启动子元件	28
		Core promoter element around -30 of transcription start
TCA-element	CCATCTTTTT	参与水杨酸响应的顺式作用元件	1
		cis-Acting element involved in salicylic acid responsiveness
TGACG/CGTCA-motif	TGACG/CGTCA	参与茉莉酸甲酯响应的顺式作用调节元件	4
		cis-Acting regulatory element involved in the MeJA-responsiveness
ARE	AAACCA	厌氧诱导的关键顺式作用调节元件	2
		cis-Acting regulatory element essential for the anaerobic induction
LTR	CCGAAA	参与低温响应的顺式作用元件	1
		cis-Acting element involved in low-temperature responsiveness
MBS	CAACTG	参与干旱诱导的MYB结合位点	2
		MYB binding site involved in drought-inducibility
circadian	CAAAGATATC	参与昼夜节律控制的顺式调控元件	1
		cis-Acting regulatory element involved in circadian control
Box 4	ATTAAT	光响应相关的保守DNA模块的一部分	1
		Part of the conserved DNA module involved in light responsiveness
G-box	CACGAC	参与光响应的顺式作用调节元件	1
		cis-Acting regulatory element involved in light responsiveness
3-AF1 binding site	TAAGAGAGGAA	光响应元件 Light responsive element	1
GT1-motif	GGTTAA	光响应元件 Light responsive element	2
Sp1	GGGCGG	光响应元件 Light responsive element	1
GA-motif	ATAGATAA	光响应元件的一部分 Part of the light responsive element	1
GTGGC-motif	GATTCTGTGGC	光响应元件的一部分 Part of the light responsive element	1

顺式作用元件	序列	功能	数量
cis-acting element	Sequence	Function	Number
CAAT-box	CCAAT	启动子和增强子区域常见的顺式作用元件	14
		Common cis-acting element in promoter and enhancer regions
TATA-box	TATAAAT/TATA	转录起始位点周围 - 30左右的核心启动子元件	28
		Core promoter element around -30 of transcription start
TCA-element	CCATCTTTTT	参与水杨酸响应的顺式作用元件	1
		cis-Acting element involved in salicylic acid responsiveness
TGACG/CGTCA-motif	TGACG/CGTCA	参与茉莉酸甲酯响应的顺式作用调节元件	4
		cis-Acting regulatory element involved in the MeJA-responsiveness
ARE	AAACCA	厌氧诱导的关键顺式作用调节元件	2
		cis-Acting regulatory element essential for the anaerobic induction
LTR	CCGAAA	参与低温响应的顺式作用元件	1
		cis-Acting element involved in low-temperature responsiveness
MBS	CAACTG	参与干旱诱导的MYB结合位点	2
		MYB binding site involved in drought-inducibility
circadian	CAAAGATATC	参与昼夜节律控制的顺式调控元件	1
		cis-Acting regulatory element involved in circadian control
Box 4	ATTAAT	光响应相关的保守DNA模块的一部分	1
		Part of the conserved DNA module involved in light responsiveness
G-box	CACGAC	参与光响应的顺式作用调节元件	1
		cis-Acting regulatory element involved in light responsiveness
3-AF1 binding site	TAAGAGAGGAA	光响应元件 Light responsive element	1
GT1-motif	GGTTAA	光响应元件 Light responsive element	2
Sp1	GGGCGG	光响应元件 Light responsive element	1
GA-motif	ATAGATAA	光响应元件的一部分 Part of the light responsive element	1
GTGGC-motif	GATTCTGTGGC	光响应元件的一部分 Part of the light responsive element	1