葡萄VlCKX4表达特性分析与转录调控预测

doi:10.3864/j.issn.0578-1752.2023.01.011

摘要/Abstract

摘要：

【目的】通过克隆细胞分裂素脱氢酶/氧化酶基因VlCKX4及其启动子，进行表达特性分析和启动子活性分析并进行转录因子预测，为深入解析VlCKX4介导细胞分裂素信号途径调控葡萄坐果的分子机制提供依据。【方法】使用生物信息学方法分析‘巨峰’葡萄(Vitis vinifera×Vitis labrusca)细胞分裂素氧化酶/脱氢酶4（VlCKX4）的序列特征；利用PCR技术克隆该基因以及它的启动子，使用实时荧光定量PCR(real-time quantitative PCR，qRT-PCR)分析VlCKX4的表达特性，GUS活性试验用于分析其启动子活性；利用PlantTFDB、CisBP等转录调控数据库对VlCKX4的转录调控关系进行预测分析，输出结果使用Gephi软件进行可视化。【结果】VlCKX4全长1 582 bp,其中包含1 566 bp的开放阅读框，编码522个氨基酸，具有家族特征的FAD结构域和细胞分裂素结合（ck-binding）结构域。qRT-PCR结果表明VlCKX4在花序中高表达，其次是叶片，在卷须中表达量最低；细胞分裂素CPPU处理后VlCKX4表达量先下降后上升，细胞分裂素抑制剂洛伐他汀(Lov)处理后VlCKX4表达量先上升后下降。顺式元件分析表明VlCKX4启动子中含有吲哚乙酸、茉莉酸甲酯等响应元件；GUS化学组织染色试验表明，VlCKX4响应这些激素的处理。VlCKX4转录调控预测分析结果显示，MYB、DOF和WRKY类转录因子对其进行转录调控，结合转录组数据和共表达关系确定WRKY20、DOF1.5和MYB59为关键候选转录因子。【结论】葡萄VlCKX4受到细胞分裂素CPPU的诱导，VlCKX4启动子受到WRKY20、DOF1.5和MYB59转录因子的调控，参与促进葡萄坐果过程。

关键词: ‘巨峰’葡萄, 细胞分裂素, VlCKX4, 启动子活性, 调控

Abstract:

【Objective】The cytokinin dehydrogenase/oxidase VlCKX4 and its promoter in grape (Vitis vinifera) were cloned to analyze the expression characteristics, promoter activity and transcription factor prediction, so as to provide a basis for the molecular mechanism that VlCKX4 involved in cytokinin-mediated berry set in grape. 【Method】The gene sequence of cytokinin oxidase/dehydrogenase 4 (VlCKX4) in Kyoho grape (Vitis vinifera × Vitis labrusca) was studied by bioinformatics method. The gene and its promoter were cloned by PCR. The expression characteristics of VlCKX4 were analyzed by real-time quantitative PCR (qRT-PCR). GUS activity assay was used to analyze the promoter activity. The transcriptional regulatory relationship of VlCKX4 was predicted by PlantTFDB, CisBP databases, and the output results were visualized by Gephi software. 【Result】The total length of VlCKX4 was 1 582 bp, which contained 1 566 bp open reading frame (ORF), encoded 522 amino acids, and had the family characteristic FAD domain and cytokinin binding (CK-binding) domain. qRT-PCR results showed that VlCKX4 was highly expressed in inflorescence and leaf, but the expression of tendril was lowest. The expression of VlCKX4 decreased first and then increased after treatment with cytokinin (CPPU), while the expression of VlCKX4 increased first and then decreased after treatment with the cytokinin inhibitor lovastatin (Lov). Cis-element analysis showed that the VlCKX4 promoter contained response elements of methyl jasmonate (MeJA) and other hormones. GUS histochemical staining showed that VlCKX4 responded to the hormones. Predictive analysis of transcriptional regulation showed that MYB, DOF and WRKY transcription factors were involved in transcriptional regulation of VlCKX4. Combined with transcriptomic expression data and coexpression relation, WRKY20, DOF1.5 and MYB59 were identified as key candidate transcription factors. 【Conclusion】 VlCKX4 was induced by CPPU and participates in the process of promoting grape berry set, during which prediction was regulated by transcription factors, such as WRKY20, DOF1.5 and MYB59.

Key words: Kyoho, Cytokinin, VlCKX4, promoter activity, regulation

李旭飞,杨盛迪,李松琦,刘海楠,裴茂松,韦同路,郭大龙,余义和. 葡萄VlCKX4表达特性分析与转录调控预测[J]. 中国农业科学, 2023, 56(1): 144-155.

LI XuFei,YANG ShengDi,LI SongQi,LIU HaiNan,PEI MaoSong,WEI TongLu,GUO DaLong,YU YiHe. Analysis of VlCKX4 Expression Characteristics and Prediction of Transcriptional Regulation in Grape[J]. Scientia Agricultura Sinica, 2023, 56(1): 144-155.

图/表 8

表1

表2

靶向VlCKX4的转录因子预测"

基因ID Gene ID	类型 Type	NR 注释 NR_annotation
Vitvi10g00581	DOF	锌指蛋白DOF1.5 DOF zinc finger protein DOF1.5 [Vitis vinifera]
Vitvi08g01336	MYB	转录因子DIVARICATA Transcription factor DIVARICATA [Vitis vinifera]
Vitvi19g00617	WRKY	可能的WRKY20转录因子 Probable WRKY transcription factor 20 [Vitis vinifera]
Vitvi18g00858	DOF	锌指蛋白DOF3.4 DOF zinc finger protein DOF3.4 [Vitis vinifera]
Vitvi16g00106	MYB	WER转录因子 Transcription factor WER [Vitis vinifera]
Vitvi12g00055	NLP	NLP3蛋白 Protein NLP3 isoform X1 [Vitis vinifera]
Vitvi04g01842	MYB	Myb类蛋白 Myb protein-like [Vitis vinifera]
Vitvi04g00854	HD-zip	HD-zip类蛋白ROC8 Homeobox-leucine zipper protein ROC8 [Vitis vinifera]
Vitvi01g00980	TCP	TCP15转录因子 Transcription factor TCP15 [Vitis vinifera]
Vitvi16g00894	MAKC	MADS-box转录因子 MADS-box transcription factor 6 isoform X1 [Vitis vinifera]
Vitvi18g01067	BPC	GAGA结合转录激活因子 GAGA-binding transcriptional activator BBR/BPC6-like [Vitis vinifera]
Vitvi15g01540	NAC	NAC结构蛋白 NAC domain-containing protein 62-like isoform X1 [Vitis vinifera]
Vitvi18g00406	MYB	MYB39转录因子 Transcription factor MYB39 [Vitis vinifera]
Vitvi01g01844	WRKY	可能的WRKY57转录因子 Probable WRKY transcription factor 57 [Vitis vinifera]
Vitvi15g01539	NAC	NTM1-like9蛋白 Protein NTM1-like 9 isoform X1 [Vitis vinifera]
Vitvi12g00121	HSF	热胁迫相关转录因子A-4b Heat stress transcription factor A-4b [Vitis vinifera]
Vitvi14g01519	TCP	转录因子TCP20 Transcription factor TCP20 [Vitis vinifera]
Vitvi15g00776	MAKC	MADS-box 3蛋白 MADS-box protein 3 [Vitis vinifera]
Vitvi03g01059	MAKC	MADS-box蛋白 MADS-box protein JOINTLESS isoform X1 [Vitis vinifera]
Vitvi14g01740	MYB	转录因子MYB98 Transcription factor MYB98 [Vitis vinifera]
Vitvi14g01398	TCP	转录因子TCP13 Transcription factor TCP13 [Vitis vinifera]
Vitvi14g01960	MYB	MYB306相关蛋白 Myb-related protein 306 [Vitis vinifera]
Vitvi15g00708	YABBY	调控边界的YABBY Axial regulator YABBY 1 isoform X2 [Vitis vinifera]
Vitvi04g01404	MAKC	MADS-box蛋白TM6 Agamous-like MADS-box protein TM6 [Vitis vinifera]
Vitvi14g00612	MYB	Myb相关蛋白Myb4 Myb-related protein Myb4 [Vitis vinifera]
Vitvi06g00414	MYB	MYB59转录因子 Transcription factor MYB59 [Vitis vinifera]
Vitvi17g00021	LEAFY	LEAFY类蛋白 LEAFY-like protein [Vitis hybrid cultivar]

表2

图1

图2

图3

表3

VlCKX4启动子顺式作用元件预测"

顺式元件 Cis-element	序列 Sequence	功能 Function	元件数量 Number
TCT-motif	TCTTAC	光调控元件 Involved in light responsiveness	1
MRE	AACCTAA	MYB结合位点参与光响应 MYB binding site involved in light responsiveness	1
AT1-motif	AATTATTTTTTATT	光调控元件 Involved in light responsiveness	1
TCA-element	CCATCTTTTT	水杨酸响应元件 Involved in salicylic acid responsiveness	1
TATC-box	TATCCCA	赤霉素响应元件 Involved in gibberellin-responsiveness	1
LTR	CCGAAA	低温响应元件 Involved in low-temperature responsiveness	1
AAGAA-motif	GAAAGAA	未知 Unknown	1
ABRE3a	TACGTG	未知 Unknown	1
WRE3	CCACCT	未知 Unknown	1
WUN-motif	AAATTTCTT	未知 Unknown	1
ABRE4	CACGTA	未知 Unknown	1
box S	AGCCACC	未知 Unknown	1
Myb	TAACTG	未知 Unknown	1
GATA-motif	GATAGGG	光调控元件 Involved in light responsiveness	2
TGA-element	AACGAC	生长素响应元件 Auxin-responsive element	2
circadian	CAAAGATATC	参与昼夜节律的元件 Components involved in the circadian rhythm	2
CGTCA-motif	CGTCA	茉莉酸甲酯诱导 Methyl jasmonate-responsive element	2
TGACG-motif	TGACG	茉莉酸甲酯诱导 Methyl jasmonate-responsive element	2
RY-element	CATGCATG	种子特异性调控 Seed specific regulation element	2
W box	TTGACC	未知 Unknown	2
as-1	TGACG	未知 Unknown	2
MYC	CATTTG	未知 Unknown	2
ABRE	ACGTG	脱落酸响应元件 In the abscisic acid responsiveness	3
Box 4	ATTAAT	光调控元件 Involved in light responsiveness	3
MYB	TAACCA/	未知 Unknown	3
MYB-like sequence	TAACCA	未知 Unknown	3
I-box	GGATAAGGTG	光调控元件 Involved in light responsiveness	5
GA-motif	ATAGATAA	光调控元件 Involved in light responsiveness	5
G-box	TACGTG	光调控元件 Involved in light responsiveness	5
STRE	AGGGG	未知 Unknown	5
GT1-motif	GGTTAAT/GGTTAA/	光调控元件 Involved in light responsiveness	6
TATA-box	TATA	核心启动元件 Core promoter element around -30 of transcription start	19
CAAT-box	CAAAT	启动子区和增强子区常见元件 Common elements in promoter and enhancer regions	25

表3

图4

图5

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基因名称 Gene name	引物 Primer
基因名称 Gene name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
FL-VlCKX4	CATGGCTAAAACTTATTCAATCCCAACAT	GTCCAGTTCTACAGCTTAAATGGAATT
Pro-VlCKX4	CCATCAACCCGACAGGATAACACC	GGACAAGTTTGCCAAAGTCCCTAGC
GUS-ProVlCKX4	GGTGGACTCCTCTTAGAATTCCCAT…ACC	TGGGCCCGGCGCGCCGAATTCGGAC…AGC
QT-VlCKX4	GACCTGTTTTATGCCGTTCTG	CATTCGCACCCACTTCACTC
Ubiqutin I	GTGGTATTATTGAGCCATCCTT	AACCTCCAATCCAGTCATCTAC

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