Screening for WRKY72-interacting Proteins Using a Yeast Two-hybrid Library Derived from Soft Rot Pathogen-induced Amorphophallus konjac

doi:10.13560/j.cnki.biotech.bull.1985.2024-0535

Abstract

Abstract:

【Objective】This study is aimed to construct a konjac cDNA library and screen for interacting proteins of the transcription factor WRKY72 in order to elucidate the molecular mechanism underlying WRKY72-mediated konjac resistance against diseases.【Method】Konjac plants at different stages of bacterial soft rot infection were used as experimental materials. Total RNA was extracted, pooled in equal amounts, and used to construct a konjac cDNA library. The bait vector containing the transcription factor WRKY72 was constructed using the homologous recombination method, and a yeast two-hybrid screen was performed to identify candidate target proteins, which were further verified individually.【Result】The yeast library titer was 1.6×10⁷ CFU/mL, and the average length of the inserted fragments was around 1 000 bp. The bait vector was verified to have no self-activation in yeast. The bait vector pGBKT7-WRKY72 was used to screen library and 41 positive interacting proteins were obtained, including transport proteins, channel proteins, heat shock proteins, chlorophyll-binding proteins, and peroxidases. Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）functional enrichment analyses were performed on these interacting proteins, and 12 of them were further verified by one-on-one yeast two-hybrid assays.【Conclusion】A high-quality konjac cDNA library was successfully constructed, and the interacting proteins of the transcription factor WRKY72 were screened, providing a theoretical basis for elucidating the mechanism of resistance against soft rot disease in konjac.

Key words: konjac soft rot, yeast two-hybrid, cDNA library, WRKY transcription factor, interacting proteins

SHEN Chuan, LI Xia, QIN Jian-feng, DUAN Long-fei, LIU Jia. Screening for WRKY72-interacting Proteins Using a Yeast Two-hybrid Library Derived from Soft Rot Pathogen-induced Amorphophallus konjac[J]. Biotechnology Bulletin, 2025, 41(1): 85-94.

Figures/Tables 9

References 32

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编号 No.	功能注释 Function annotation		序列号 Accession
1	平衡核苷酸转运蛋白1样蛋白	Equilibrative nucleotide transporter 1-like	AH13G08570.1
2	蛋白酶体亚基 β-6型	Proteasome subunit beta type-6	XM_021751824.2
3	水通道蛋白 PIP2-6	Aquaporin PIP2-6	XM_010921795.3
4	叶绿素 a-b 结合蛋白	Chlorophyll a-b binding protein	XM_009391104.2
5	类热休克蛋白质	Class I heat shock protein-like	XM_037569548.1
6	小泛素相关修饰物1	Small ubiquitin-related modifier 1	XM_009414468.2
7	磷酸核酮糖3-异构酶	Ribulose-phosphate 3-epimerase	XM_020248600.1
8	TVP38/TMEm64家族膜蛋白sr0305	TVP38/TMEM64 family membrane protein slr0305-like	XM_039134159.1
9	小分子热休克蛋白	Small heat shock protein	XM_008813654.3
10	蛋白质转运蛋白Sec61亚基	Protein transport protein Sec61 subunit gamma	XM_037612338.1
11	液泡蛋白分选相关蛋白13C	Vacuolar protein sorting-associated protein 13C	XM_039279221.1
12	光系统 I 反应中心亚基	Photosystem I reaction center subunit II	XM_009402563.2
13	延伸因子1-a	Elongation factor 1-alpha	XM_031625179.2
14	类 γ-谷氨酰水解酶2	Gamma-glutamyl hydrolase 2-like	XM_059806763.1
15	31kD 核糖核蛋白	31 kD ribonucleoprotein	XM_008791202.4
16	类似苹果酸脱氢酶	Malate dehydrogenase, glyoxysomal-like	XM_008780993.4
17	铁氧化还原蛋白	Ferredoxin	XM_051344602.1
18	叶绿素 a-b 结合蛋白4	Chlorophyll a-b binding protein 4	XM_020255110.1
19	脂磷酸磷酸酶 δ	Lipid phosphate phosphatase delta	XM_010936202.3
20	硫氧还蛋白 H 型	Thioredoxin H-type	XM_062288332.1
21	LHCII III 型叶绿素 a-b 结合蛋白	Chlorophyll a-b binding protein of LHCII type III	XM_039974480.1
22	核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基	Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit	AF034479.1
23	大核糖体亚基蛋白 eL39	Large ribosomal subunit protein eL39	XM_062364610.1
24	LHCII 类1型叶绿素 a-b 结合蛋白	Chlorophyll a-b binding protein of LHCII type 1-like	XM_009391104.2
25	叶绿素 a-b 结合蛋白8	Chlorophyll a-b binding protein 8	XM_057956264.1
26	镁-原卟啉 IX 单甲酯氧化环化酶	Magnesium-protoporphyrin IX monomethyl ester Oxidative cyclase	XM_018819649.1
27	类赖氨酸组氨酸转运蛋白	Lysine histidine transporter 1-like	XM_020737698.1
28	乙酰鸟氨酸脱乙酰基酶	Acetylornithine deacetylase	XM_010935186.3
29	过氧化物酶21	Peroxidase 21	XM_009393918.1
30	核转录因子 Y 亚基 A-7	Nuclear transcription factor Y subunit A-7	XM_021736266.2
31	谷氨酸 tRNA 连接酶	Glutamate-tRNA ligase	XM_058220292.1
32	Rubisco激活酶（rca1）	Rubisco activase（rca1）	AF338237.1
33	光系统 I 叶绿素 a/b 结合蛋白2	Photosystem I chlorophyll a/b-binding protein 2	XM_042535174.1
34	叶绿素a-b结合蛋白 CP29.1	Chlorophyll a-b binding protein CP29.1	XM_062333380.1
35	热休克蛋白	Heat shock protein	XM_010930652.3
36	丝氨酸/苏氨酸蛋白磷酸酶2A	Serine/threonine protein phosphatase 2A	XM_017844298.3
37	鸟氨酸氨基转移酶	Ornithine transcarbamylase	XM_043855639.1
38	60S 核糖体蛋白质 L34-like	60S ribosomal protein L34-like	XM_039037995.1
39	60S 核糖体蛋白质 L18-2-like	60S ribosomal protein L18-2-like	XM_039120108.1
40	谷氨酰胺合成酶	Glutamine synthetase	XM_010940494.3
41	琥珀酸脱氢酶	Succinate dehydrogenase	XM_020232587.1

编号 No.	功能注释 Function annotation		序列号 Accession
1	平衡核苷酸转运蛋白1样蛋白	Equilibrative nucleotide transporter 1-like	AH13G08570.1
2	蛋白酶体亚基 β-6型	Proteasome subunit beta type-6	XM_021751824.2
3	水通道蛋白 PIP2-6	Aquaporin PIP2-6	XM_010921795.3
4	叶绿素 a-b 结合蛋白	Chlorophyll a-b binding protein	XM_009391104.2
5	类热休克蛋白质	Class I heat shock protein-like	XM_037569548.1
6	小泛素相关修饰物1	Small ubiquitin-related modifier 1	XM_009414468.2
7	磷酸核酮糖3-异构酶	Ribulose-phosphate 3-epimerase	XM_020248600.1
8	TVP38/TMEm64家族膜蛋白sr0305	TVP38/TMEM64 family membrane protein slr0305-like	XM_039134159.1
9	小分子热休克蛋白	Small heat shock protein	XM_008813654.3
10	蛋白质转运蛋白Sec61亚基	Protein transport protein Sec61 subunit gamma	XM_037612338.1
11	液泡蛋白分选相关蛋白13C	Vacuolar protein sorting-associated protein 13C	XM_039279221.1
12	光系统 I 反应中心亚基	Photosystem I reaction center subunit II	XM_009402563.2
13	延伸因子1-a	Elongation factor 1-alpha	XM_031625179.2
14	类 γ-谷氨酰水解酶2	Gamma-glutamyl hydrolase 2-like	XM_059806763.1
15	31kD 核糖核蛋白	31 kD ribonucleoprotein	XM_008791202.4
16	类似苹果酸脱氢酶	Malate dehydrogenase, glyoxysomal-like	XM_008780993.4
17	铁氧化还原蛋白	Ferredoxin	XM_051344602.1
18	叶绿素 a-b 结合蛋白4	Chlorophyll a-b binding protein 4	XM_020255110.1
19	脂磷酸磷酸酶 δ	Lipid phosphate phosphatase delta	XM_010936202.3
20	硫氧还蛋白 H 型	Thioredoxin H-type	XM_062288332.1
21	LHCII III 型叶绿素 a-b 结合蛋白	Chlorophyll a-b binding protein of LHCII type III	XM_039974480.1
22	核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基	Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit	AF034479.1
23	大核糖体亚基蛋白 eL39	Large ribosomal subunit protein eL39	XM_062364610.1
24	LHCII 类1型叶绿素 a-b 结合蛋白	Chlorophyll a-b binding protein of LHCII type 1-like	XM_009391104.2
25	叶绿素 a-b 结合蛋白8	Chlorophyll a-b binding protein 8	XM_057956264.1
26	镁-原卟啉 IX 单甲酯氧化环化酶	Magnesium-protoporphyrin IX monomethyl ester Oxidative cyclase	XM_018819649.1
27	类赖氨酸组氨酸转运蛋白	Lysine histidine transporter 1-like	XM_020737698.1
28	乙酰鸟氨酸脱乙酰基酶	Acetylornithine deacetylase	XM_010935186.3
29	过氧化物酶21	Peroxidase 21	XM_009393918.1
30	核转录因子 Y 亚基 A-7	Nuclear transcription factor Y subunit A-7	XM_021736266.2
31	谷氨酸 tRNA 连接酶	Glutamate-tRNA ligase	XM_058220292.1
32	Rubisco激活酶（rca1）	Rubisco activase（rca1）	AF338237.1
33	光系统 I 叶绿素 a/b 结合蛋白2	Photosystem I chlorophyll a/b-binding protein 2	XM_042535174.1
34	叶绿素a-b结合蛋白 CP29.1	Chlorophyll a-b binding protein CP29.1	XM_062333380.1
35	热休克蛋白	Heat shock protein	XM_010930652.3
36	丝氨酸/苏氨酸蛋白磷酸酶2A	Serine/threonine protein phosphatase 2A	XM_017844298.3
37	鸟氨酸氨基转移酶	Ornithine transcarbamylase	XM_043855639.1
38	60S 核糖体蛋白质 L34-like	60S ribosomal protein L34-like	XM_039037995.1
39	60S 核糖体蛋白质 L18-2-like	60S ribosomal protein L18-2-like	XM_039120108.1
40	谷氨酰胺合成酶	Glutamine synthetase	XM_010940494.3
41	琥珀酸脱氢酶	Succinate dehydrogenase	XM_020232587.1