葡萄脱落酸受体VvPYL4互作蛋白的筛选及互作蛋白基因表达

doi:10.13560/j.cnki.biotech.bull.1985.2024-0964

摘要/Abstract

摘要：

目的 ABA受体PYRl/PYLs/RCARs在ABA信号转导通路中起着重要作用。通过酵母双杂交技术筛选葡萄VvPYL4的互作蛋白，探究VvPYL4在葡萄应答霜霉病菌胁迫信号通路中的作用。方法以霜霉病菌侵染‘贝达’葡萄叶片为材料，构建cDNA文库；构建诱饵表达载体pGBKT7-VvPYL4，通过酵母双杂交技术从cDNA文库中筛选与VvPYL4相互作用的蛋白；通过实时荧光定量PCR分析候选互作蛋白基因在葡萄霜霉病菌诱导下的表达模式，并通过双分子荧光互补技术进行互作蛋白的验证。结果构建的酵母双杂交cDNA文库库容为7.16×10⁷ CFU/mL，重组率100%，插入片段大小在1 000 bp左右。成功构建诱饵表达载体pGBKT7-VvPYL4，且在酵母细胞中无自激活活性。诱饵载体与酵母双杂交文库共转酵母AH109菌株后，经多次筛库、测序、BLAST比对和回转验证，最终获得53个候选互作蛋白，这些蛋白涉及信号转导、植物生长发育及环境胁迫响应等多个方面。基于实时荧光定量PCR分析，编码4个蛋白的基因均受葡萄霜霉病菌诱导表达。通过双分子荧光互补试验发现，在共转pSPYCE-PP2C24和pSPYNE-PYL4表达载体的本氏烟草叶片中可观察到强烈的黄色荧光信号，表明PYL4与PP2C24蛋白之间能够发生相互作用。结论成功构建霜霉病菌侵染葡萄叶片的cDNA文库，并筛选出53个与VvPYL4相互作用的候选蛋白，其中4个编码蛋白的基因均响应葡萄霜霉病菌的胁迫诱导，验证了VvPYL4与PP2C24蛋白之间存在相互作用关系。

关键词: 葡萄霜霉病, 脱落酸受体PYL4, cDNA文库筛选, 酵母双杂交系统, 双分子荧光互补, 互作蛋白, 蛋白磷酸酶2C

Abstract:

Objective The ABA receptors PYRl/PYLs/RCARs play an important role in the ABA signal transduction pathway. The interaction proteins of VvPYL4 were screened by yeast two-hybrid technique to explore signaling pathway of the VvPYL4 responding to Plasmopara viticola infection in grape. Method The cDNA library was constructed using the Beta grape leaves infected with P. viticola. The bait expression vector pGBKT7-VvPYL4 was constructed to facilitate the identification of proteins that interact with VvPYL4 from the cDNA library by yeast two-hybrid technology. The expression patterns of candidate interacting protein genes induced by P. viticola were analyzed using real-time PCR and the interactions of the proteins were verified using bimolecular fluorescence complementation technology. Result The results titer of the cDNA library was 7.16×10⁷ CFU/mL, the recombination rate was 100%, and the average length encoded by the inserted cDNA was around 1 000 bp. The bait expression vector pGBKT7-VvPYL4 was successfully constructed. Result showed that there was no autoactivation activity in the yeast cells. The cDNA library plasmid and bait vector pGBKT7-VvPYL4 were co-transformed into yeast AH109 strain. After several screening, sequencing, BLAST alignment and rotation verification, 53 candidate proteins were obtained, which were involved in signal transduction, plant growth and development, and environmental stress response, respectively. Based on real-time PCR analysis, the expressions of genes encoding four proteins were induced by P. viticola. During a bimolecular fluorescence complementation assay, the robust yellow fluorescence signals were detected in the leaves of T. benthamiana that were co-transformed with the pSPYCE-PP2C24 and pSPYNE-PYL4 expression vectors, suggesting an interaction between the PYL4 and PP2C24 proteins. Conclusion A cDNA library is constructed from grapevine leaves infected with P. viticola, leading to the identification of 53 candidate proteins that interact with VvPYL4. Among these, the genes encoding four proteins are found to respond to stress induced by P. viticola. Furthermore, the interaction between VvPYL4 and the PP2C24 protein is confirmed.

Key words: grape downy mildew, abscisic acid receptor PYL4, screening of cDNA library, yeast two-hybrid system, bimolecular fluorescence complementation, interaction protein, PP2C

刘丽, 王辉, 关天舒, 李柏宏, 于舒怡. 葡萄脱落酸受体VvPYL4互作蛋白的筛选及互作蛋白基因表达[J]. 生物技术通报, 2025, 41(4): 188-197.

LIU Li, WANG Hui, GUAN Tian-shu, LI Bai-hong, YU Shu-yi. Screening the Interacting Protein of Abscisic Acid Receptor VvPYL4 and the Gene Expression of the Interacting Protein in Grape[J]. Biotechnology Bulletin, 2025, 41(4): 188-197.

图/表 9

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基因 Gene	正向引物序列 Forward primer sequence （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）
VvMYB3R-3	CTTTCCCAATACTCCTTCCA	CCCAGTTCTTTCTTGTTCATC
VvPP2C24	CTCGTGTTCCTCTTTCGTTC	CATTCTTCCAACCTGACTCC
VvASR1	CCTACTCCGACACCACCTAC	GTGCTTCTCCTCCTTCCTG
VvPOD6	TGCAGTGAAACTTCTTCAGC	CATCCACCCTTCCATACTTC
EF1a	GAACTGGGTGCTTGATAGGC	ACCAAAATATCCGGAGTAAAAGA

基因 Gene	正向引物序列 Forward primer sequence （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）
VvMYB3R-3	CTTTCCCAATACTCCTTCCA	CCCAGTTCTTTCTTGTTCATC
VvPP2C24	CTCGTGTTCCTCTTTCGTTC	CATTCTTCCAACCTGACTCC
VvASR1	CCTACTCCGACACCACCTAC	GTGCTTCTCCTCCTTCCTG
VvPOD6	TGCAGTGAAACTTCTTCAGC	CATCCACCCTTCCATACTTC
EF1a	GAACTGGGTGCTTGATAGGC	ACCAAAATATCCGGAGTAAAAGA

编号 No.	酵母克隆编号 Code of yeast clone	基因编号 Gene ID	功能注释 Function annotation	筛选次数 Number of screenings
1	BD-VvPYL4-4	LOC117926668	Thaumatin-like protein 1	1
2	BD-VvPYL4-6	LOC100245034	Hypersensitive-induced reaction 1 protein	1
3	BD-VvPYL4-10	LOC117928201	Small heat shock protein	1
4	BD-VvPYL4-12	LOC100256181	Transcription factor MYB3R-3	1
5	BD-VvPYL4-13	LOC100260476	Pathogenesis-related protein PR-4	2
6	BD-VvPYL4-16	LOC117913055	Glutamate-glyoxylate aminotransferase 2	1
7	BD-VvPYL4-18	LOC100246378	Glyceraldehyde-3-phosphate dehydrogenase A	1
8	BD-VvPYL4-19	LOC100248362	Mitochondrial outer membrane protein porin of 36 kDa	1
9	BD-VvPYL4-20	LOC100257132	Glycine rich protein family	1
10	BD-VvPYL4-21	LOC100245393	Chlorophyll A-B binding protein	1
11	BD-VvPYL4-22	LOC100268135	Chaperone protein dnaJ A6	1
12	BD-VvPYL4-23	LOC100249605	Expansin-A13	1
13	BD-VvPYL4-25	LOC117905970	Thioredoxin-like protein CDSP32	1
14	BD-VvPYL4-26	LOC100255251	Protein phosphatase 2C 24	2
15	BD-VvPYL4-27	LOC100258879	Cytochrome b6-f complex iron-sulfur subunit 1	3
16	BD-VvPYL4-29	LOC100233124	RD22-like protein	3
17	BD-VvPYL4-30	LOC100254430	Protein DEHYDRATION-INDUCED 19 homolog 3	2
18	BD-VvPYL4-31	LOC100251062	ATP synthase delta chain	1
19	BD-VvPYL4-33	LOC117916106	Ribulose bisphosphate carboxylase/oxygenase activase	2
20	BD-VvPYL4-34	LOC117919543	Probable galactinol-sucrose galactosyltransferase 2	1
21	BD-VvPYL4-35	LOC100526774	Cytochrome P450	1
22	BD-VvPYL4-36	LOC100852851	Fructose-bisphosphate aldolase 1	1
23	BD-VvPYL4-37	LOC100247997	Anthocyanidin 3-O-glucosyltransferase 5	1
24	BD-VvPYL4-38	LOC117932928	Ethylene-responsive transcription factor 5-like	1
25	BD-VvPYL4-39	LOC117906248	Purine permease 1-like	1
26	BD-VvPYL4-40	LOC100254009	Peroxidase 6	1
27	BD-VvPYL4-41	LOC100240756	Endo-1, 3; 1, 4-beta-D-glucanase-like	1
28	BD-VvPYL4-42	LOC100241982	Phospholipase D delta	1
29	BD-VvPYL4-44	LOC100264642	Probable 3-hydroxyisobutyrate dehydrogenase-like 1	1
30	BD-VvPYL4-45	LOC100245816	Methyl-CpG-binding domain-containing protein 9	1
31	BD-VvPYL4-47	LOC100255748	Uncharacterized	1
32	BD-VvPYL4-50	LOC100267253	Metallothionein	1
33	BD-VvPYL4-52	LOC100260811	Oxygen-evolving enhancer protein 1	1
34	BD-VvPYL4-53	LOC100253703	Rac-like GTP-binding protein RAC1	1
35	BD-VvPYL4-54	LOC100250281	Abscisic stress-ripening protein 1-like	1
36	BD-VvPYL4-55	LOC117911472	Ferredoxin--nitrite reductase	1
37	BD-VvPYL4-58	LOC100853165	Catalase-related immune-responsive	1
38	BD-VvPYL4-60	LOC100267892	20 kD chaperonin	1
39	BD-VvPYL4-61	LOC117905125	Protein P21-like	4
40	BD-VvPYL4-62	LOC100247939	Cinnamoyl-CoA reductase 1	1
41	BD-VvPYL4-64	LOC100252685	Leucine Rich repeat	1
42	BD-VvPYL4-65	LOC100264042	Tim17/Tim22/Tim23/Pmp24 family	1
43	BD-VvPYL4-69	LOC100249137	Beta carbonic anhydrase 1	1
44	BD-VvPYL4-74	LOC117928074	Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit A-like	1
45	BD-VvPYL4-76	LOC100855103	Domain of unknown function	1
46	BD-VvPYL4-77	LOC100245544	Bifunctional monothiol glutaredoxin-S16	1
47	BD-VvPYL4-78	LOC117907066	Elongation factor 1-gamma	1
48	BD-VvPYL4-79	LOC100264196	Cysteine synthase	3
49	BD-VvPYL4-83	LOC117915980	Elongation factor 1-alpha	1
50	BD-VvPYL4-85	LOC100267386	Aldehyde dehydrogenase family	1
51	BD-VvPYL4-86	LOC117904623	Chaperone protein ClpB1	1
52	BD-VvPYL4-87	LOC117924474	Probable galactinol-sucrose galactosyltransferase 6	1
53	BD-VvPYL4-88	LOC100243967	Photosystem II 10 kD polypeptide PsbR	1

编号 No.	酵母克隆编号 Code of yeast clone	基因编号 Gene ID	功能注释 Function annotation	筛选次数 Number of screenings
1	BD-VvPYL4-4	LOC117926668	Thaumatin-like protein 1	1
2	BD-VvPYL4-6	LOC100245034	Hypersensitive-induced reaction 1 protein	1
3	BD-VvPYL4-10	LOC117928201	Small heat shock protein	1
4	BD-VvPYL4-12	LOC100256181	Transcription factor MYB3R-3	1
5	BD-VvPYL4-13	LOC100260476	Pathogenesis-related protein PR-4	2
6	BD-VvPYL4-16	LOC117913055	Glutamate-glyoxylate aminotransferase 2	1
7	BD-VvPYL4-18	LOC100246378	Glyceraldehyde-3-phosphate dehydrogenase A	1
8	BD-VvPYL4-19	LOC100248362	Mitochondrial outer membrane protein porin of 36 kDa	1
9	BD-VvPYL4-20	LOC100257132	Glycine rich protein family	1
10	BD-VvPYL4-21	LOC100245393	Chlorophyll A-B binding protein	1
11	BD-VvPYL4-22	LOC100268135	Chaperone protein dnaJ A6	1
12	BD-VvPYL4-23	LOC100249605	Expansin-A13	1
13	BD-VvPYL4-25	LOC117905970	Thioredoxin-like protein CDSP32	1
14	BD-VvPYL4-26	LOC100255251	Protein phosphatase 2C 24	2
15	BD-VvPYL4-27	LOC100258879	Cytochrome b6-f complex iron-sulfur subunit 1	3
16	BD-VvPYL4-29	LOC100233124	RD22-like protein	3
17	BD-VvPYL4-30	LOC100254430	Protein DEHYDRATION-INDUCED 19 homolog 3	2
18	BD-VvPYL4-31	LOC100251062	ATP synthase delta chain	1
19	BD-VvPYL4-33	LOC117916106	Ribulose bisphosphate carboxylase/oxygenase activase	2
20	BD-VvPYL4-34	LOC117919543	Probable galactinol-sucrose galactosyltransferase 2	1
21	BD-VvPYL4-35	LOC100526774	Cytochrome P450	1
22	BD-VvPYL4-36	LOC100852851	Fructose-bisphosphate aldolase 1	1
23	BD-VvPYL4-37	LOC100247997	Anthocyanidin 3-O-glucosyltransferase 5	1
24	BD-VvPYL4-38	LOC117932928	Ethylene-responsive transcription factor 5-like	1
25	BD-VvPYL4-39	LOC117906248	Purine permease 1-like	1
26	BD-VvPYL4-40	LOC100254009	Peroxidase 6	1
27	BD-VvPYL4-41	LOC100240756	Endo-1, 3; 1, 4-beta-D-glucanase-like	1
28	BD-VvPYL4-42	LOC100241982	Phospholipase D delta	1
29	BD-VvPYL4-44	LOC100264642	Probable 3-hydroxyisobutyrate dehydrogenase-like 1	1
30	BD-VvPYL4-45	LOC100245816	Methyl-CpG-binding domain-containing protein 9	1
31	BD-VvPYL4-47	LOC100255748	Uncharacterized	1
32	BD-VvPYL4-50	LOC100267253	Metallothionein	1
33	BD-VvPYL4-52	LOC100260811	Oxygen-evolving enhancer protein 1	1
34	BD-VvPYL4-53	LOC100253703	Rac-like GTP-binding protein RAC1	1
35	BD-VvPYL4-54	LOC100250281	Abscisic stress-ripening protein 1-like	1
36	BD-VvPYL4-55	LOC117911472	Ferredoxin--nitrite reductase	1
37	BD-VvPYL4-58	LOC100853165	Catalase-related immune-responsive	1
38	BD-VvPYL4-60	LOC100267892	20 kD chaperonin	1
39	BD-VvPYL4-61	LOC117905125	Protein P21-like	4
40	BD-VvPYL4-62	LOC100247939	Cinnamoyl-CoA reductase 1	1
41	BD-VvPYL4-64	LOC100252685	Leucine Rich repeat	1
42	BD-VvPYL4-65	LOC100264042	Tim17/Tim22/Tim23/Pmp24 family	1
43	BD-VvPYL4-69	LOC100249137	Beta carbonic anhydrase 1	1
44	BD-VvPYL4-74	LOC117928074	Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit A-like	1
45	BD-VvPYL4-76	LOC100855103	Domain of unknown function	1
46	BD-VvPYL4-77	LOC100245544	Bifunctional monothiol glutaredoxin-S16	1
47	BD-VvPYL4-78	LOC117907066	Elongation factor 1-gamma	1
48	BD-VvPYL4-79	LOC100264196	Cysteine synthase	3
49	BD-VvPYL4-83	LOC117915980	Elongation factor 1-alpha	1
50	BD-VvPYL4-85	LOC100267386	Aldehyde dehydrogenase family	1
51	BD-VvPYL4-86	LOC117904623	Chaperone protein ClpB1	1
52	BD-VvPYL4-87	LOC117924474	Probable galactinol-sucrose galactosyltransferase 6	1
53	BD-VvPYL4-88	LOC100243967	Photosystem II 10 kD polypeptide PsbR	1

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