Screening the Interacting Protein of Abscisic Acid Receptor VvPYL4 and the Gene Expression of the Interacting Protein in Grape

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

Abstract

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

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.

Figures/Tables 9

References 42

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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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