葡萄转录因子VlWRKY14和VlTCP5响应外源CPPU并调控VlIPT12的表达

doi:10.16420/j.issn.0513-353x.2024-0761

摘要/Abstract

摘要：

细胞分裂素CPPU[forchlorfenuron，N-（2-chloro-4-pyridinyl）-N-phenylurea]处理葡萄花序后能显著提高葡萄的坐果率，但分子机制尚不清楚。为阐明异戊烯基转移酶基因VlIPT12在CPPU处理后的表达及其特定转录因子调控的分子机制，从‘巨峰’葡萄中克隆得到VlIPT12基因，对其进行生物信息分析、表达特性与转录调控分析。结果显示，VlIPT12基因全长984 bp，具有IPT家族特有的ATP/ADP结合位点（P-loop motif），定位在细胞核。VlIPT12基因在幼果中表达量最高，其次是花序和卷须，在叶片中表达量最低。CPPU处理葡萄花序后，VlIPT12基因表达量显著低于对照。VlIPT12启动子响应水杨酸和茉莉酸甲酯的处理。VlWRKY14和VlTCP5作为VlIPT12的关键转录因子，在CPPU处理后的表达模式与VlIPT12一致，并且可以与VlIPT12启动子关键位点（P1、P2、P4）结合促进其表达。综上所述，CPPU处理葡萄花序后，VlIPT12表达量下降，转录因子VlWRKY14和VlTCP5可以与VlIPT12启动子的关键位点结合，正调控VlIPT12在幼果中的表达。

关键词: 葡萄, 坐果, 细胞分裂素, VlIPT12, 转录调控

Abstract:

Treatment with the cytokinin CPPU[forchlorfenuron，N-（2-chloro-4-pyridinyl）-N- phenylurea]significantly improves the fruit setting rate in grapevines，but the underlying molecular mechanisms remain unclear. To elucidate the expression of the isopentenyl transferase gene VlIPT12 after CPPU treatment and the molecular mechanism of its regulation by specific transcription factors，in this study，the VlIPT12 was cloned from‘Kyoho’grapes，and bioinformatics analysis，expression characteristics，and transcriptional regulation analysis were performed. The results showed that the full length of the VlIPT12 was 984 bp，containing a conserved ATP/ADP binding site（P-loop motif）specific to the IPT family and localizing in the nucleus. The expression level of the VlIPT12 gene was the highest in young fruits，followed by that in flowers and tendrils，and was the lowest in leaves. After the grape inflorescences were treated with CPPU，the expression level of VlIPT12 gene was significantly lower than that of the control. The VlIPT12 promoter responded to the treatments of salicylic acid and methyl jasmonate. VlWRKY14 and VlTCP5，as the key transcription factors of VlIPT12，had the same expression pattern as VlIPT12 after CPPU treatment and could bind to the key sites（P1，P2，P4）of the VlIPT12 promoter to promote its expression. In conclusion，after the grape inflorescences were treated with CPPU，the expression level of VlIPT12 decreased，and the transcription factors VlWRKY14 and VlTCP5 could bind to the key sites of the VlIPT12 promoter to positively regulate the expression of VlIPT12 in young fruits.

Key words: grape, berry set, cytokinin, VlIPT12, transcriptional regulation

赵亚如, 王磊磊, 孔冠焯, 李泓, 郑锦涛, 岳伊涵, 史巧芳, 赵晓春, 余义和. 葡萄转录因子VlWRKY14和VlTCP5响应外源CPPU并调控VlIPT12的表达[J]. 园艺学报, 2025, 52(10): 2567-2581.

ZHAO Yaru, WANG Leilei, KONG Guanzhuo, LI Hong, ZHENG Jintao, YUE Yihan, SHI Qiaofang, ZHAO Xiaochun, YU Yihe. Transcription Factors VlWRKY14 and VlTCP5 Respond to Exogenous CPPU and Regulate the Expression of VlIPT12 in Grape[J]. Acta Horticulturae Sinica, 2025, 52(10): 2567-2581.

图/表 10

表1 在本研究中用到的引物

Table 1 The primer used in this study

用途 Usage	引物名称 Primer name	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
基因克隆 Gene clone	FL-VlIPT12	GGACCTCACCATGGATGTCTCC	CTAGTGGGTTGCTGCTAGGGCA
	Pro-VlIPT12	GGGCAATTTGGGTCTGAAGTGCT	GTAGAATCAGAGATGCAGCAACCG
	FL-VlWRKY14	GTGATCCTTCAGCTGGTTTCCCA	CCAAGTTTATGCCAGGACCCTTG
	FL-VlTCP5	CCTGCACGCTGGACCATCAAGAA	CCATGGCGAAGCATACTATCTGGC
GUS活性验证 GUS activity verification	GUS-ProVlIPT12	TGGGCCCGGCGCGCCAAGCTTG GGCAATTTGGGTCTGAAGTGCT	GGTGGACTCCTCTTAGAATTCGT AGAATCAGAGATGCAGCAACCG
亚细胞定位 Subcellular localization	YFP-VlIPT12	ATGGGATCTACTAGTGAATTCGGACCTCACCATGGATGTCTCC	GGGGGTACCGTCGACGGATCCCTAGTGGGTTGCTGCTAGGGCA
DLR验证 DLR verification	LUC-VlIPT12	GTCGACGGTATCGATAAGCTTGGACCTCACCATGGATGTCTCC	CGCTCTAGAACTAGTGGATCC CTAGTGGGTTGCTGCTAGGGCA
Y1H验证 Y1H verification	TCP5-IPT12-TFBD1	GAGGAACCATTCTTCAAAGTGGG	CCCAAATTGCCCATGATTATGGC
	TCP5-IPT12-TFBD2	GATTTGATACGAGTCATCTGG	ACCGACATCTGATTCCTATCC
	TCP5-IPT12-TFBD3	GGATATCAACATCACCCTGCCC	TTAGCTTGGAGTGGTGGCCC
	WRKY14-IPT12-TFBD1	GTACTATTGACCTTACGCGC	GCCTACGAGTAAATGATAGTC
	WRKY14-IPT12-TFBD2	CTTAATAGATAGAAACCCTCCC	CTACCAATCATATGTCTCAAGA
	WRKY14-IPT12-TFBD3	GGCCACCACTCCAAGCTAAGC	GGGTCAAGGACCTCTGCTCAAC
	AD-EcoRI-QWRKY14	GCCATGGAGGCCAGTGAATTCGTGATCCTTCAGCTGGTTTCCCA	CAGCTCGAGCTCGATGGATCC CCAAGTTTATGCCAGGACCCTTG
	AD-EcoRI-QTCP5	GCCATGGAGGCCAGTGAATTCCCTGCACGCTGGACCATCAAGAA	CAGCTCGAGCTCGATGGATCCCCATGGCGAAGCATACTATCTGGC

图1 VlIPT12的序列分析和亚细胞定位 A：VlIPT12的PCR扩增；B：葡萄与其他植物的IPT蛋白氨基酸多序列对比；C：VlIPT12的亚细胞定位

Fig. 1 Sequence analysis and subcellular localization of VlIPT12 A：PCR amplification of VlIPT12；B：Comparison of multiple amino acid sequences of IPT protein from grape and other plants；C：Subcellular localization of VlIPT12

图 2 CPPU处理葡萄后VlIPT12的表达模式 A：幼果中，数据为平均值 ± SD，t检验（** P < 0.01，*** P < 0.001）。下同。B：不同组织中；列上方的不同小写字母表示不同组织之间基因表达水平显著差异，数据为平均值 ± SD，单因素方差分析（P < 0.05）。下同

Fig. 2 Expression patterns of VlIPT12 in grapes after CPPU treatment A：In young grape berry，data are presented as means ± SD，analyzed by t-test（** P < 0.01，*** P < 0.001）. The same below. B：In different tissues；different lowercase letters above the columns indicate significant differences in gene expression levels among tissues，data are presented as means ± SD，analyzed by one-way ANOVA（P < 0.05）. The same below

表2 VlIPT12启动子序列中的主要顺式作用元件

Table 2 The major cis-acting elements of VlIPT12 promoter

顺式作用元件 cis-Acting elements	序列 Sequence	功能 Function	元件数量 Number
TGACG-motif	TGACG	茉莉酸甲酯响应元件 MeJA responsive element	1
GT1-motif	GGTTAA	光调控元件 Light responsive element	2
GATA-motif	GATAGGG/GATAGGA	光调控元件 Light responsive element	2
Sp1	GGGCGG	光调控元件 Light responsive element	1
TCT-motif	TCTTAC	光调控元件 Light responsive element	1
TGA-element	AACGAC	生长素响应元件 Auxin responsive element	1
MBS	CAACTG	参与干旱诱导的调控元件Drought-inducibility responsive element	2
TCA-element	CCATCTTTTT	水杨酸响应元件 Salicylic acid responsive element	1
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件 MeJA responsive element	1
W-box	TGACC	未知 Unknown	2
CAT-box	GCCACT	分生组织表达相关调控元件 cis-Acting regulatory element related to meristem expression	2
GC-motif	CCCCCG	参与缺氧特异性诱导的增强子元件 Enhancer-like element involved in anoxic specific inducibility	1
ARE	AAACCA	参与厌氧特异性诱导的增强子元件 cis-Acting regulatory element essential for the anaerobic induction	1
MYB	CAACCA/TAACCA/CAACTG	未知 Unknown	4
AAGAA-motif	GAAAGAA	未知 Unknown	1
TATA-box	TATA	核心启动元件 Core promoter element	36
CAAT-box	CAAT/CCAAT/CAAAT	启动子区和增强子区常见元件 Common cis-acting element in promoter and enhancer regions	42

图3 VlIPT12启动子中激素响应的顺式作用元件

Fig. 3 cis-Acting elements responsive to hormones in the VlIPT12 promoter

图4 VlIPT12启动子GUS活性分析 GUS空载构建体作为阴性对照，CaMC35S::GUS构建体作为阳性对照

Fig. 4 Analysis of GUS activity of VlIPT12 promoter Empty GUS vector as a negative control and the CaMC35S::GUS construct as a positive control

图5 VlIPT12互作的转录因子预测 A：TFDB和CIS-BP数据库预测VlIPT12互作的转录因子韦恩图；B：VlIPT12的靶向关系图；C：调控VlIPT12的转录因子富集；D：CPPU处理后VlIPT12的潜在互作转录因子热图

Fig. 5 Prediction of transcription factors interacting with VlIPT12 A：Veen diagram of transcription factors predicted to interact with VlIPT12 by TFDB and CIS-BP database；B：Targeting relationship map of VlIPT12；C：Enrichment analysis of transcription factors regulating VlIPT12；D：Heatmap of potential interacting transcription factors of VlIPT12 after CPPU treatment

图6 VlWRKY14和VlTCP5在外源CPPU处理后的表达量

Fig. 6 Expression levels of VlWRKY14 and VlTCP5 after exogenous CPPU treatment

图7 双荧光素酶LUC/REN活性检测

Fig. 7 Dual luciferase LUC/REN activity assay

图8 VlWRKY14、VlTCP5与VlIPT12酵母单杂交互作验证 A：VlIPT12启动子分段示意图；三角形表示转录因子结合位点的位置，P片段代表转录因子的预测结合位点；B：酵母单杂交显示VlWRKY14/VlTCP5与VlIPT12启动子片段结合

Fig. 8 Yeast one-hybrid interaction assay of VlWRKY14，VlTCP5 with VlIPT12 A：Schematic diagram of VlIPT12 promoter segmentation. Triangles indicate the positions of transcription factor binding sites，and fragment P represents the predicted binding site of transcription factors；B：Yeast one-hybrid assay shows that VlWRKY14 and VlTCP5 bind to VlIPT12 promoter fragments

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