Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (1): 144-155.doi: 10.3864/j.issn.0578-1752.2023.01.011

• HORTICULTURE • Previous Articles     Next Articles

Analysis of VlCKX4 Expression Characteristics and Prediction of Transcriptional Regulation in Grape

LI XuFei(),YANG ShengDi,LI SongQi,LIU HaiNan,PEI MaoSong,WEI TongLu,GUO DaLong,YU YiHe()   

  1. College of Horticulture and Plant Protection, Henan University of Science and Technology/Henan Engineering Technology Research Center of Quality Regulation and Controlling of Horticultural Plants, Luoyang 471023, Henan
  • Received:2022-03-19 Accepted:2022-07-19 Online:2023-01-01 Published:2023-01-17
  • Contact: YiHe YU E-mail:lixufei8023 @163.com;yuyihe@haust.edu.cn

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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

Table 1

The primer used in this study"

基因名称
Gene name		 引物 Primer
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

Table 2

The transcription factor prediction of VlCKX4 gene"

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]

Fig. 1

PCR amplified result of VlCKX4 and its promoter Marker: DL2000"

Fig. 2

VlCKX4 structure diagram and phylogenetic tree"

Fig. 3

Expression of VlCKX4 in grape tissues after hormone treatment *: P<0.05; **: P<0.01; ***: P<0.001"

Table 3

The cis-acting elements prediction of VlCKX4 gene promoter"

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

Fig. 4

GUS histochemical staining after plant growth regulator treatment"

Fig. 5

Prediction of transcription factors that target VlCKX4 A: Enrichment of transcription factor that regulate VlCKX4 gene. The size indicates the number of transcription factors, and the color represent the effect of regulatory relations; B: Mapping of VlCKX4 targeting. The line between circles represents a targeting relationship. The same color line represents transcription factors of the same type, and the number of transcription factors less than 1 is uniformly represented by gray. The strength of co-expression relationship is reflected in the node size, the larger the node, the stronger the co-expression relationship between the transcription factor and VlCKX4"

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