Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (11): 2846-2860.doi: 10.16420/j.issn.0513-353x.2024-0889
• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles
LIU Miaomiao, YAO Jin, ZOU Fuyan, CHU Yanyan, and WANG Xiping*(
)
Received:2025-03-22
Revised:2025-07-02
Online:2025-11-26
Published:2025-11-26
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and WANG Xiping
LIU Miaomiao, YAO Jin, ZOU Fuyan, CHU Yanyan, and WANG Xiping. Screening and Validation of Interacting Proteins of the Grape Ovule Development Transcription Factor VvDDF2[J]. Acta Horticulturae Sinica, 2025, 52(11): 2846-2860.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2024-0889
| 用途 Purpose | 引物名称 Primer name | 上游序列(5′-3′) Forward sequence | 下游序列(5′-3′) Reverse sequence |
|---|---|---|---|
| 转录自激活验证 Transcriptional self-activation validation | VvDDF2-BD | atggccatggaggccgaattcATGGACTTCTTCAATCAATTTTGTAATT | cgctgcaggtcgacggatccTCAAATTGAGTAACTCCATAGTGACATATC |
| VvDDF2-D1-BD | atgg ccatggaggccgaattcATGGACTTCTTCAATCAATTTTGTAATT | cgctgcaggtcgacggatccCTGAGGCCGGAACGCCTC | |
| 酵母双杂交点对点回复验证 Yeast two-hybrid point-to-point response validation | VvEXO70A1-AD | gccatggaggccagtgaattcATGGGCGTAGCAGTAGGCATG | cagctcgagctcgatggatccTCGCTTGGCTTCATTCAAGGTT |
| VvBBP-AD | gccatggaggccagtgaattcATGGCTGTGGGAAGAGGCAGTG | cagctcgagctcgatggatccAGTAGCAGTTACTGCTATCTTCATT | |
| BiFC实验 BiFC assay | VvDDF2-NE | ggcgcgccactagtggatccATGGACTTCTTCAATCAATTTTGTAATT | catcccgggagcggtaccAATTGAGTAACTCCATAGTGACATATC |
| VvEXO70A1-CE | ggcgcgccactagtggatccATGGGCGTAGCAGTAGGCATG | catcccgggagcggtaccTCGCTTGGCTTCATTCAAGGTT | |
| Split-Luc实验 Split-Luc assay | VvDDF2-N-Luc | tcggtacccgggatccATGGACTTCTTCAATCAATTTTGTAATT | acgagatctggtcgacAATTGAGTAACTCCATAGTGACATATC |
| VvEXO70A1-C-Luc | acgcgtcccggggcggtaccATGGGCGTAGCAGTAGGCATG | ta cgaacgaaagctctgcagTTATCGCTTGGCTTCATTCAAGGTT | |
| 基因表达分析 Gene expression analysis | VvActin-qPCR | GATTCTGGTGATGGTGTGAGT | GACAATTTCCCGTTCAGCAGT |
| VvEXO70A1-qPCR | CGTCAGGCAGAGGGTAAAG | AGGAGGGTCGCAGTGAGTT |
Table 1 Primer sequences used in this study
| 用途 Purpose | 引物名称 Primer name | 上游序列(5′-3′) Forward sequence | 下游序列(5′-3′) Reverse sequence |
|---|---|---|---|
| 转录自激活验证 Transcriptional self-activation validation | VvDDF2-BD | atggccatggaggccgaattcATGGACTTCTTCAATCAATTTTGTAATT | cgctgcaggtcgacggatccTCAAATTGAGTAACTCCATAGTGACATATC |
| VvDDF2-D1-BD | atgg ccatggaggccgaattcATGGACTTCTTCAATCAATTTTGTAATT | cgctgcaggtcgacggatccCTGAGGCCGGAACGCCTC | |
| 酵母双杂交点对点回复验证 Yeast two-hybrid point-to-point response validation | VvEXO70A1-AD | gccatggaggccagtgaattcATGGGCGTAGCAGTAGGCATG | cagctcgagctcgatggatccTCGCTTGGCTTCATTCAAGGTT |
| VvBBP-AD | gccatggaggccagtgaattcATGGCTGTGGGAAGAGGCAGTG | cagctcgagctcgatggatccAGTAGCAGTTACTGCTATCTTCATT | |
| BiFC实验 BiFC assay | VvDDF2-NE | ggcgcgccactagtggatccATGGACTTCTTCAATCAATTTTGTAATT | catcccgggagcggtaccAATTGAGTAACTCCATAGTGACATATC |
| VvEXO70A1-CE | ggcgcgccactagtggatccATGGGCGTAGCAGTAGGCATG | catcccgggagcggtaccTCGCTTGGCTTCATTCAAGGTT | |
| Split-Luc实验 Split-Luc assay | VvDDF2-N-Luc | tcggtacccgggatccATGGACTTCTTCAATCAATTTTGTAATT | acgagatctggtcgacAATTGAGTAACTCCATAGTGACATATC |
| VvEXO70A1-C-Luc | acgcgtcccggggcggtaccATGGGCGTAGCAGTAGGCATG | ta cgaacgaaagctctgcagTTATCGCTTGGCTTCATTCAAGGTT | |
| 基因表达分析 Gene expression analysis | VvActin-qPCR | GATTCTGGTGATGGTGTGAGT | GACAATTTCCCGTTCAGCAGT |
| VvEXO70A1-qPCR | CGTCAGGCAGAGGGTAAAG | AGGAGGGTCGCAGTGAGTT |
Fig. 1 Amplification of VvDDF2 full-length and deletion fragment(VvDDF2-D1)(A),construction of bait vector(B),toxicity detection(C)and analysis of transcriptional self-activating activity(D) -T:SD/-Trp;-T/X:SD/-Trp/X-α-gal;-T/X/A:SD/-Trp + X-α-Gal/AbA
| 编号 Number | 登录号 Sequence ID | 蛋白名称 Protein name | 功能注释 Annotation | 参考文献 Reference |
|---|---|---|---|---|
| 1 | XM_010652827.3 | 腺苷酸激酶同工酶6同源物 Adenylate kinase isoenzyme 6 homolog [Vitis vinifera(wine grape)] | 腺苷酸激酶(ADK)是一种能量代谢的关键酶,维持细胞能量动态平衡,在植物中参与调节生物产量和逆境应答等过程 Adenylate kinase(ADK)is a key enzyme in energy metabolism that maintains the dynamic balance of cellular energy,and is involved in regulating biological production and response to adversity in plants | Gong et al., |
| 2 | XM_010656680.3 | 反式高尔基体网状结构定位的SYP41相互作用蛋白1 trans-Golgi network- localized SYP41-interacting protein 1[V. vinifera(wine grape)] | 拟南芥SYP4家族定位于TGN(trans-Golgi Network),参与种子贮藏蛋白前体运输途径,调控植物生长发育 The SYP4 family in Arabidopsis is localized in the TGN (trans-Golgi Network)and is involved in the seed storage protein precursor transport pathway,regulating plant growth and development | Uemura et al., |
| 3 | XM_003631207.4 | 2-亚甲基-呋喃-3-酮还原酶 2-Methylene-furan-3-one reductase [V. vinifera(wine grape)] | 一种乙醇脱氢酶(ADH),与果实成熟过程中醇的形成密切相关,GmADH1可能参与了大豆质核互作雄性不育(CMS)的育性调控 A type of alcohol dehydrogenase(ADH),closely related to the formation of alcohols during fruit ripening,GmADH1 may be involved in the regulation of cytoplasmic-nuclear male sterility(CMS)in soybean | 丁先龙, |
续 | ||||
| 编号 Number | 登录号 Sequence ID | 蛋白名称 Protein name | 功能注释 Annotation | 参考文献 Reference |
| 4 | XM_002266352.3 | DCD结构域蛋白NRP-B DCD domain-containing protein NRP-B [V. vinifera(wine grape)] | DCD/NRP蛋白在植物激素的响应、胚胎发育和病原体或臭氧导致的程序性细胞死亡中起作用 DCD/NRP proteins play a role in plant hormone response,embryonic development and programmed cell death caused by pathogens or ozone | Tenhaken et al., |
| 5 | XM_002285094.5 | 4-羟基-3-甲基丁-2-烯基二磷酸还原酶(HDR) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ferredoxin),chloroplastic [V. vinifera(wine grape)] | 与质体发育有关,定位于叶绿体,参与植物异戊烯基二磷酸(IPP)和胆固醇生物合成,是萜烯类物质生物合成的重要限速酶 Associated with plastid development,localized in chloroplasts,involved in the biosynthesis of isopentenyl diphosphate(IPP)and cholesterol in plants,important rate limiting enzyme for terpenoid biosynthesis | 陈天池 等, |
| 6 | XM_002275951.5 | 碱性蓝铜蛋白 Basic blue protein[V. vinifera (wine grape)] | 一种植物铜氧还蛋白,属于蓝铜蛋白家族的成员,参与生殖授粉、器官形成和胁迫响应 A plant copper oxyreticulin protein,a member of the blue copper protein family,involved in reproductive pollination,organ formation and stress response | Dong et al., |
| 7 | XM_002268074.5 | 胞外囊复合体亚基 Exocyst complex component EXO70A1[V. vinifera(wine grape)] | 参与胞质分裂、细胞极性生长和形态建成、花粉萌发和花粉管的极性伸长、种皮细胞壁的形成和PIN蛋白的再循环和极性定位、植物免疫反应和自噬等过程 Involved in cytoplasmic division,polar growth and morphogenesis of cells,pollen germination and polar elongation of pollen tubes,formation of seed coat cell wall,recirculation and polar localization of PIN proteins,plant immune response and autophagy | Hala et al., |
| 8 | XM_002285852.4 | BTB/POZ结构域蛋白 BTB/POZ domain-containing protein At4g08455[V. vinifera (wine grape)] | 与植物激素信号转导、抗逆性、蛋白质泛素化、细胞骨架构成、次生代谢产物合成等有关 Related to plant hormone signal transduction,stress resistance,protein ubiquitination,cytoskeleton composition and secondary metabolite synthesis | Julian et al., |
| 9 | XM_059743404.1 | 胺氧化酶 Amine oxidase [copper- containing] gamma 1[V. vinifera (wine grape)] | 参与植物多胺合成代谢通路 Involved in plant polyamine synthesis metabolic pathway | Fortes et al., |
| 10 | XM_010653238.3 | 羟甲基戊二酰辅酶A裂解酶 Hydroxymethylglutaryl-CoA lyase,mitochondrial[V. vinifera(wine grape)] | 一种线粒体蛋白,在大豆中调节豆荚色素沉着和开裂 A mitochondrial proteins that regulates pod pigmentation and cracking in soybean | Lyu et al., |
| 11 | XM_010651472.2 | 脂肪酶 Lipase [V. vinifera(wine grape)] | 参与植物种子中的油脂代谢过程,影响种子发育;拟南芥GDSL脂肪酶基因在胚乳中表达并且调节胚胎的发育 Involvement in lipid metabolism in plant seeds that affects seed development. The GDSL lipase gene in Arabidopsis is expressed in endosperm and regulates embryo development | Clauss et al., |
| 12 | XM_002283173.4 | 谷胱甘肽转移酶(GST) Glutathione S-transferase F9[V. vinifera(wine grape)] | 植物体中广泛存在的一种多功能酶,在植物的生长发育、次生代谢和抵御非生物胁迫发挥作用 A multifunctional enzyme widely found in plants that plays a role in plant growth and development,secondary metabolism and resistance to abiotic stresses | Rezaei et al., |
| 13 | XM_059740061.1 | 核糖体大亚基蛋白 Large ribosomal subunit protein bL21m[V. vinifera(wine grape)] | 参与蛋白质合成,调控油菜自交不亲和反应 Involvement in protein synthesis and regulation of self-incompatibility response in oilseed rape | Khan, |
| 14 | XM_010657812.3 | 醛脱氢酶7家族成员 Aldehyde dehydrogenase family 7 member [V. vinifera(wine grape)] | 醛脱氢酶(ALDH)参与植物对生物及非生物的胁迫反应以及对生长发育的调节 Aldehyde dehydrogenase(ALDH)is involved in plant responses to biotic and abiotic stresses and regulation of growth and development | 张玉成 等, |
Table 2 Candidate interacting proteins obtained from yeast two-hybrid library screening
| 编号 Number | 登录号 Sequence ID | 蛋白名称 Protein name | 功能注释 Annotation | 参考文献 Reference |
|---|---|---|---|---|
| 1 | XM_010652827.3 | 腺苷酸激酶同工酶6同源物 Adenylate kinase isoenzyme 6 homolog [Vitis vinifera(wine grape)] | 腺苷酸激酶(ADK)是一种能量代谢的关键酶,维持细胞能量动态平衡,在植物中参与调节生物产量和逆境应答等过程 Adenylate kinase(ADK)is a key enzyme in energy metabolism that maintains the dynamic balance of cellular energy,and is involved in regulating biological production and response to adversity in plants | Gong et al., |
| 2 | XM_010656680.3 | 反式高尔基体网状结构定位的SYP41相互作用蛋白1 trans-Golgi network- localized SYP41-interacting protein 1[V. vinifera(wine grape)] | 拟南芥SYP4家族定位于TGN(trans-Golgi Network),参与种子贮藏蛋白前体运输途径,调控植物生长发育 The SYP4 family in Arabidopsis is localized in the TGN (trans-Golgi Network)and is involved in the seed storage protein precursor transport pathway,regulating plant growth and development | Uemura et al., |
| 3 | XM_003631207.4 | 2-亚甲基-呋喃-3-酮还原酶 2-Methylene-furan-3-one reductase [V. vinifera(wine grape)] | 一种乙醇脱氢酶(ADH),与果实成熟过程中醇的形成密切相关,GmADH1可能参与了大豆质核互作雄性不育(CMS)的育性调控 A type of alcohol dehydrogenase(ADH),closely related to the formation of alcohols during fruit ripening,GmADH1 may be involved in the regulation of cytoplasmic-nuclear male sterility(CMS)in soybean | 丁先龙, |
续 | ||||
| 编号 Number | 登录号 Sequence ID | 蛋白名称 Protein name | 功能注释 Annotation | 参考文献 Reference |
| 4 | XM_002266352.3 | DCD结构域蛋白NRP-B DCD domain-containing protein NRP-B [V. vinifera(wine grape)] | DCD/NRP蛋白在植物激素的响应、胚胎发育和病原体或臭氧导致的程序性细胞死亡中起作用 DCD/NRP proteins play a role in plant hormone response,embryonic development and programmed cell death caused by pathogens or ozone | Tenhaken et al., |
| 5 | XM_002285094.5 | 4-羟基-3-甲基丁-2-烯基二磷酸还原酶(HDR) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ferredoxin),chloroplastic [V. vinifera(wine grape)] | 与质体发育有关,定位于叶绿体,参与植物异戊烯基二磷酸(IPP)和胆固醇生物合成,是萜烯类物质生物合成的重要限速酶 Associated with plastid development,localized in chloroplasts,involved in the biosynthesis of isopentenyl diphosphate(IPP)and cholesterol in plants,important rate limiting enzyme for terpenoid biosynthesis | 陈天池 等, |
| 6 | XM_002275951.5 | 碱性蓝铜蛋白 Basic blue protein[V. vinifera (wine grape)] | 一种植物铜氧还蛋白,属于蓝铜蛋白家族的成员,参与生殖授粉、器官形成和胁迫响应 A plant copper oxyreticulin protein,a member of the blue copper protein family,involved in reproductive pollination,organ formation and stress response | Dong et al., |
| 7 | XM_002268074.5 | 胞外囊复合体亚基 Exocyst complex component EXO70A1[V. vinifera(wine grape)] | 参与胞质分裂、细胞极性生长和形态建成、花粉萌发和花粉管的极性伸长、种皮细胞壁的形成和PIN蛋白的再循环和极性定位、植物免疫反应和自噬等过程 Involved in cytoplasmic division,polar growth and morphogenesis of cells,pollen germination and polar elongation of pollen tubes,formation of seed coat cell wall,recirculation and polar localization of PIN proteins,plant immune response and autophagy | Hala et al., |
| 8 | XM_002285852.4 | BTB/POZ结构域蛋白 BTB/POZ domain-containing protein At4g08455[V. vinifera (wine grape)] | 与植物激素信号转导、抗逆性、蛋白质泛素化、细胞骨架构成、次生代谢产物合成等有关 Related to plant hormone signal transduction,stress resistance,protein ubiquitination,cytoskeleton composition and secondary metabolite synthesis | Julian et al., |
| 9 | XM_059743404.1 | 胺氧化酶 Amine oxidase [copper- containing] gamma 1[V. vinifera (wine grape)] | 参与植物多胺合成代谢通路 Involved in plant polyamine synthesis metabolic pathway | Fortes et al., |
| 10 | XM_010653238.3 | 羟甲基戊二酰辅酶A裂解酶 Hydroxymethylglutaryl-CoA lyase,mitochondrial[V. vinifera(wine grape)] | 一种线粒体蛋白,在大豆中调节豆荚色素沉着和开裂 A mitochondrial proteins that regulates pod pigmentation and cracking in soybean | Lyu et al., |
| 11 | XM_010651472.2 | 脂肪酶 Lipase [V. vinifera(wine grape)] | 参与植物种子中的油脂代谢过程,影响种子发育;拟南芥GDSL脂肪酶基因在胚乳中表达并且调节胚胎的发育 Involvement in lipid metabolism in plant seeds that affects seed development. The GDSL lipase gene in Arabidopsis is expressed in endosperm and regulates embryo development | Clauss et al., |
| 12 | XM_002283173.4 | 谷胱甘肽转移酶(GST) Glutathione S-transferase F9[V. vinifera(wine grape)] | 植物体中广泛存在的一种多功能酶,在植物的生长发育、次生代谢和抵御非生物胁迫发挥作用 A multifunctional enzyme widely found in plants that plays a role in plant growth and development,secondary metabolism and resistance to abiotic stresses | Rezaei et al., |
| 13 | XM_059740061.1 | 核糖体大亚基蛋白 Large ribosomal subunit protein bL21m[V. vinifera(wine grape)] | 参与蛋白质合成,调控油菜自交不亲和反应 Involvement in protein synthesis and regulation of self-incompatibility response in oilseed rape | Khan, |
| 14 | XM_010657812.3 | 醛脱氢酶7家族成员 Aldehyde dehydrogenase family 7 member [V. vinifera(wine grape)] | 醛脱氢酶(ALDH)参与植物对生物及非生物的胁迫反应以及对生长发育的调节 Aldehyde dehydrogenase(ALDH)is involved in plant responses to biotic and abiotic stresses and regulation of growth and development | 张玉成 等, |
Fig. 3 Yeast two-hybrid analysis of the interaction between VvDDF2 and candidate proteins A:Construction of candidate gene prey vectors;B:Point-to-point response verification of yeast two-hybrid. DDO:SD/-Leu/-Trp;QDO:SD/-Ade/-His/-Leu/-Trp;QDO/X/A:SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA
Fig. 4 BiFC detects the interaction between VvDDF2 and VvEXO70A1 A:Construction of BiFC related vectors;B:Observation of fluorescence signals in tobacco leaf cells
Fig. 5 Split-Luc to detect the interaction between VvDDF2 and VvEXO70A1 A:Construction of Split-Luc related vectors;B:Tobacco leaf fluorescence detection
Fig. 6 Semi-quantitative PCR(A)and RT-qPCR(B)analysis of the expression pattern of VvEXO70A1 during the critical period of ovule development in seeded and seedless grapes The error line represents the standard deviation(SD),and ** represents the significance of difference(P < 0.01)
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