葡萄胚珠发育转录因子VvDDF2互作蛋白的筛选与验证

doi:10.16420/j.issn.0513-353x.2024-0889

摘要/Abstract

摘要：

为探究葡萄转录因子VvDDF2参与胚珠发育与无核性状形成的调控机制，构建了酵母双杂交诱饵载体pGBKT7-VvDDF2，验证了VvDDF2具有转录自激活活性，且活性区域位于C端。利用酵母双杂交技术，从葡萄胚珠发育cDNA酵母文库中筛选获得VvDDF2的14个候选互作蛋白，包括脂肪酶、胺氧化酶、胞外囊复合体亚基、核糖体大亚基蛋白等多种酶和蛋白，主要参与细胞分裂生长和形态建成、胚胎发育、植物激素信号转导、育性调控以及各类物质的生物合成和代谢反应，在植物种子发育过程中发挥重要的生物学功能。通过酵母双杂交、双分子荧光互补和分裂荧光素酶互补实验进一步验证了VvDDF2与胞外囊复合体亚基VvEXO70A1之间存在互作关系。RT-qPCR分析表明，VvEXO70A1在有核及无核葡萄胚珠发育过程中的表达水平存在显著差异。综上，VvDDF2与VvEXO70A1两者可能通过蛋白相互作用形成复合物协同调控葡萄胚珠发育进程。

关键词: 葡萄, 无核机制, 胚珠发育, VvDDF2, 蛋白互作, VvEXO70A1

Abstract:

In order to investigate the regulatory mechanism of the grape transcription factor VvDDF2 in ovule development and seedless traits formation，the yeast two-hybrid bait vector pGBKT7-VvDDF2 was constructed. It was verified that VvDDF2 had transcriptional self-activation activity and the active region was located at the C terminal. Using yeast two-hybrid system（Y2H），14 candidate interacting proteins of VvDDF2 were screened from the yeast cDNA yeast library related to the development of grape ovule，including lipase，amine oxidase，exocyst complex component，large ribosomal subunit protein and other enzymes and proteins，which are mainly involved in cell division，growth，and morphogenesis，embryonic development，plant hormone signal transduction and fertility regulation，as well as biosynthesis and metabolic reactions of various substances，and play an important biological function in the process of plant seed development. The interaction between VvDDF2 and VvEXO70A1 was further verified by yeast two-hybrid system（Y2H），bimolecular fluorescence complementation（BiFC）and luciferase reporter system（Split-Luc）. RT-qPCR analysis showed that the expression level of VvEXO70A1 during the development of grape ovule were significantly different between seeded and seedless grapes. In summary，VvDDF2 and VvEXO70A1 may form a complex through protein interaction to synergistically regulate the development process of grape ovule.

Key words: grape, seedless mechanism, ovule development, VvDDF2, protein interaction, VvEXO70A1

刘苗苗, 姚锦, 邹福艳, 楚言言, 王西平. 葡萄胚珠发育转录因子VvDDF2互作蛋白的筛选与验证[J]. 园艺学报, 2025, 52(11): 2846-2860.

LIU Miaomiao, YAO Jin, ZOU Fuyan, CHU Yanyan, 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.

图/表 8

参考文献 40

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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
转录自激活验证 Transcriptional self-activation validation	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
BiFC实验 BiFC assay	VvEXO70A1-CE	ggcgcgccactagtggatccATGGGCGTAGCAGTAGGCATG	catcccgggagcggtaccTCGCTTGGCTTCATTCAAGGTT
Split-Luc实验 Split-Luc assay	VvDDF2-N-Luc	tcggtacccgggatccATGGACTTCTTCAATCAATTTTGTAATT	acgagatctggtcgacAATTGAGTAACTCCATAGTGACATATC
Split-Luc实验 Split-Luc assay	VvEXO70A1-C-Luc	acgcgtcccggggcggtaccATGGGCGTAGCAGTAGGCATG	ta cgaacgaaagctctgcagTTATCGCTTGGCTTCATTCAAGGTT
基因表达分析 Gene expression analysis	VvActin-qPCR	GATTCTGGTGATGGTGTGAGT	GACAATTTCCCGTTCAGCAGT
基因表达分析 Gene expression analysis	VvEXO70A1-qPCR	CGTCAGGCAGAGGGTAAAG	AGGAGGGTCGCAGTGAGTT

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
转录自激活验证 Transcriptional self-activation validation	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
BiFC实验 BiFC assay	VvEXO70A1-CE	ggcgcgccactagtggatccATGGGCGTAGCAGTAGGCATG	catcccgggagcggtaccTCGCTTGGCTTCATTCAAGGTT
Split-Luc实验 Split-Luc assay	VvDDF2-N-Luc	tcggtacccgggatccATGGACTTCTTCAATCAATTTTGTAATT	acgagatctggtcgacAATTGAGTAACTCCATAGTGACATATC
Split-Luc实验 Split-Luc assay	VvEXO70A1-C-Luc	acgcgtcccggggcggtaccATGGGCGTAGCAGTAGGCATG	ta cgaacgaaagctctgcagTTATCGCTTGGCTTCATTCAAGGTT
基因表达分析 Gene expression analysis	VvActin-qPCR	GATTCTGGTGATGGTGTGAGT	GACAATTTCCCGTTCAGCAGT
基因表达分析 Gene expression analysis	VvEXO70A1-qPCR	CGTCAGGCAGAGGGTAAAG	AGGAGGGTCGCAGTGAGTT

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.,2010
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.,2012
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,2017
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.,2005
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,2023
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.,2005
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.,2008
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.,2019
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.,2019
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.,2023
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.,2011
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.,2013
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,2020
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,2012

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.,2010
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.,2012
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,2017
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.,2005
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,2023
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.,2005
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.,2008
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.,2019
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.,2019
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.,2023
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.,2011
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.,2013
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,2020
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,2012