毛竹PheNAC4互作蛋白的筛选与分析

doi:10.16420/j.issn.0513-353x.2024-0755

摘要/Abstract

摘要：

前期试验表明毛竹（Phyllostachys edulis）PheNAC4基因在盐胁迫后下调表达，且与胁迫相关NAC基因的蛋白序列具有较高的相似性，过表达PheNAC4降低转基因拟南芥对盐胁迫的耐受性。本研究中以PheNAC4为诱饵，采用酵母双杂交系统筛选与其相互作用的蛋白，并进一步探究其调控机制。试验结果显示，PheNAC4对酵母细胞无毒性，具有自激活活性。添加1 000 ng · L^-1金担子素A抑制剂能抑制PheNAC4的自激活活性。通过筛选和序列分析，得到36个与PheNAC4互作的候选蛋白，这些候选蛋白可能与毛竹对干旱、盐胁迫的响应以及衰老调控密切相关。为验证相互作用，选择2个与胁迫相关的候选蛋白（PheR40C1和PheF3HL），通过酵母双杂交回转和双分子荧光互补试验（bimolecular fluorescence complementation，BiFC），验证PheNAC4能够与PheR40C1和PheF3HL在酵母细胞和植物细胞中形成异源二聚体。对盐胁迫后PheR40C1和PheF3HL基因的表达模式进行分析，结果表明PheR40C1的表达与PheNAC4在胁迫后的12 h具有一致性，但PheF3HL与PheNAC4呈相反的表达模式，以上结果表明PheNAC4可能通过与这两个基因互作参与毛竹的盐胁迫响应。

关键词: 毛竹, 蛋白互作, 胁迫, PheNAC4, 酵母双杂交

Abstract:

Previous experiments have shown that the PheNAC4 gene of Phyllostachys edulis（moso bamboo）is down-regulated after salt stress，and its protein sequence is highly similar to other stress-related NAC genes. Overexpression of the PheNAC4 gene reduces the tolerance of transgenic Arabidopsis thaliana to salt stress. To further investigate its regulatory mechanism，PheNAC4 was used as bait to screen for interacting proteins in this study. The results indicated that PheNAC4 is non-toxic to yeast Y2H Gold cells，although it exhibits self-activation activity in the yeast two-hybrid assay. The self-activation activity of PheNAC4 can be inhibited by adding 1 000 ng · L^-1 Aureobasidin A（AbA）inhibitor. In the yeast two-hybrid experiment，36 candidate proteins interacting with PheNAC4 were identified through screening and sequence analysis. These candidate proteins are likely involved in the response of moso bamboo to drought and salt stress，as well as in senescence regulation. To validate these interactions，two stress-related candidate proteins，PheR40C1 and PheF3HL，were selected for further investigation. The yeast two-hybrid assay and bimolecular fluorescence complementation（BiFC）experiments were conducted. These experiments confirmed that PheNAC4 forms heterodimers with PheR40C1 and PheF3HL in both yeast and plant cells. Analysis of the expression patterns of the PheR40C1 and PheF3HL genes after salt stress showed that PheR40C1 was down-regulated in the first 12 hour，exhibiting a similar expression pattern to that of the PheNAC4 gene. In contrast，PheF3HL displayed an opposite expression pattern compared to PheNAC4. These results suggest that PheNAC4 may play a crucial role in the salt stress response of moso bamboo through interactions with PheR40C1 and PheF3HL.

Key words: Phyllostachys edulis, interactive protein, stress, PheNAC4, Yeast two-hybrid system

谢丽华, 姚鹏强, 刘佳雪, 王哲, 朱楠楠, 程世平, 程占超. 毛竹PheNAC4互作蛋白的筛选与分析[J]. 园艺学报, 2025, 52(10): 2677-2690.

XIE Lihua, YAO Pengqiang, LIU Jiaxue, WANG Zhe, ZHU Nannan, CHENG Shiping, CHENG Zhanchao. Screening and Analysis of Phyllostachys edulis PheNAC4 Interacting Proteins by Yeast Two-Hybrid System[J]. Acta Horticulturae Sinica, 2025, 52(10): 2677-2690.

图/表 12

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引物名称 Primer name	引物序列（5′-3′） Primers sequences	用途 Function
RT-PheNAC4-F	ACTACCCAGGCCATGGCCT	相对表达水平分析 Relative expression level analysis
RT-PheNAC4-R	AGGCAGGCTTGTGCTCAGCT
RT-TIP41-F	AAAATCATTGTAGGCCATTGTCG
RT-TIP41-R	ACTAAATTAAGCCAGCGGGAGTG
RT-PheR40C1-F	CACCAGCCCACCTTCAAGAT
RT-PheR40C1-R	AGGGAGTGTTTGATGGCGAG
RT-PheF3HL-F	AGGGTGGATATAGAGGGTATCAAAGAG
RT-PheF3HL-R	GTTCAATAGGCGTGTAGCAATCAATC
2300-PheNAC4-F	CACCATTGCCACCTCTCACT	转基因拟南芥检测 Transgenic Arabidopsis detection
2300-R	GCCTCTTCGCTATTACGCCA	转基因拟南芥检测 Transgenic Arabidopsis detection
BD-PheNAC4-F	GGCCGAATTCCCGGGGATGTCCATGAGCTTCTTGAGCAT	诱饵载体构建 Construction of bait vector
BD-PheNAC4-R	GCCGCTGCAGGTCGACGCTAGAAGGGATTCATCCAAGTAGAG	诱饵载体构建 Construction of bait vector
AD-PheR40C1-F	TGGCCATGGAGGCCAGTGAATTCATGTTCAACTTCGGGCACCA	候选互作蛋白基因序列AD载体构建Construction of AD vector for candidate interacting protein gene sequence
AD-PheR40C1-R	CTACGATTCATCTGCAGCTCGAGTTACCAGGGGACGATCTTCCA
AD- PheF3HL-F	TGGCCATGGAGGCCAGTGAATTCATGGCCATGGCGGCGGCG
AD- PheF3HL-R	CTACGATTCATCTGCAGCTCGAGCTAAGTCAAGTCGGAGAGAAATTGT
PheNAC4-F(YN)	ACAAGTTTGTACAAAAAAATGTCCATGAGCTTCTTGAG	BiFC融合载体构建 Construction of BiFC vector
PheNAC4-R(YN)	CACCACTTTGTACAAGAAGAAGGGATTCATCCAAGTAGA
PheF3HL-F(YC)	ACAAGTTTGTACAAAAAAATGGGTTCCAGCTTCAAGGC
PheF3HL-R(YC)	CACCACTTTGTACAAGAACATGACGAAGTTGGTCGAGA
PheR40C1-F(YC)	ACAAGTTTGTACAAAAAAATGTTCAACTTCGGGCACCA
PheR40C1-R(YC)	CACCACTTTGTACAAGAACCAGGGGACGATCTTCCA

引物名称 Primer name	引物序列（5′-3′） Primers sequences	用途 Function
RT-PheNAC4-F	ACTACCCAGGCCATGGCCT	相对表达水平分析 Relative expression level analysis
RT-PheNAC4-R	AGGCAGGCTTGTGCTCAGCT
RT-TIP41-F	AAAATCATTGTAGGCCATTGTCG
RT-TIP41-R	ACTAAATTAAGCCAGCGGGAGTG
RT-PheR40C1-F	CACCAGCCCACCTTCAAGAT
RT-PheR40C1-R	AGGGAGTGTTTGATGGCGAG
RT-PheF3HL-F	AGGGTGGATATAGAGGGTATCAAAGAG
RT-PheF3HL-R	GTTCAATAGGCGTGTAGCAATCAATC
2300-PheNAC4-F	CACCATTGCCACCTCTCACT	转基因拟南芥检测 Transgenic Arabidopsis detection
2300-R	GCCTCTTCGCTATTACGCCA	转基因拟南芥检测 Transgenic Arabidopsis detection
BD-PheNAC4-F	GGCCGAATTCCCGGGGATGTCCATGAGCTTCTTGAGCAT	诱饵载体构建 Construction of bait vector
BD-PheNAC4-R	GCCGCTGCAGGTCGACGCTAGAAGGGATTCATCCAAGTAGAG	诱饵载体构建 Construction of bait vector
AD-PheR40C1-F	TGGCCATGGAGGCCAGTGAATTCATGTTCAACTTCGGGCACCA	候选互作蛋白基因序列AD载体构建Construction of AD vector for candidate interacting protein gene sequence
AD-PheR40C1-R	CTACGATTCATCTGCAGCTCGAGTTACCAGGGGACGATCTTCCA
AD- PheF3HL-F	TGGCCATGGAGGCCAGTGAATTCATGGCCATGGCGGCGGCG
AD- PheF3HL-R	CTACGATTCATCTGCAGCTCGAGCTAAGTCAAGTCGGAGAGAAATTGT
PheNAC4-F(YN)	ACAAGTTTGTACAAAAAAATGTCCATGAGCTTCTTGAG	BiFC融合载体构建 Construction of BiFC vector
PheNAC4-R(YN)	CACCACTTTGTACAAGAAGAAGGGATTCATCCAAGTAGA
PheF3HL-F(YC)	ACAAGTTTGTACAAAAAAATGGGTTCCAGCTTCAAGGC
PheF3HL-R(YC)	CACCACTTTGTACAAGAACATGACGAAGTTGGTCGAGA
PheR40C1-F(YC)	ACAAGTTTGTACAAAAAAATGTTCAACTTCGGGCACCA
PheR40C1-R(YC)	CACCACTTTGTACAAGAACCAGGGGACGATCTTCCA

顺式作用元件 cis-Acting element		功能 Function	数量 Number
激素 Hormone	TGA-element	生长素响应元件 Auxin response element	1
	ABRE	脱落酸响应元件 Abscisic acid response element	3
	CGTCA-motif	茉莉酸甲酯响应元件 MeJA response element	5
	GARE-motif	赤霉素响应元件 Gibberellin response element	1
胁迫 Stress	LTR	低温响应元件 Low temperature response element	2
	GC-motif	特异缺氧响应元件 Anoxic specific response element	1
	ARE	厌氧诱导响应元件 Anaerobic induction response element	2
	TC-rich repeats	防御和胁迫响应元件 Defense and stress response element	1
发育 Development	CAT-box	分生组织表达响应元件 Meristem expression response element	1
	GCN4_motif	分生组织特异性激活元件 cis-Regulatory element involved in endosperm expression	4
	MSA-like	参与细胞周期调控的顺式作用元件 cis-Acting element involved in cell cycle regulation	1
	O2-site	玉米醇溶蛋白代谢调控中的顺式调控元件 cis-Acting regulatory element involved in zein metabolism regulation	1

顺式作用元件 cis-Acting element		功能 Function	数量 Number
激素 Hormone	TGA-element	生长素响应元件 Auxin response element	1
	ABRE	脱落酸响应元件 Abscisic acid response element	3
	CGTCA-motif	茉莉酸甲酯响应元件 MeJA response element	5
	GARE-motif	赤霉素响应元件 Gibberellin response element	1
胁迫 Stress	LTR	低温响应元件 Low temperature response element	2
	GC-motif	特异缺氧响应元件 Anoxic specific response element	1
	ARE	厌氧诱导响应元件 Anaerobic induction response element	2
	TC-rich repeats	防御和胁迫响应元件 Defense and stress response element	1
发育 Development	CAT-box	分生组织表达响应元件 Meristem expression response element	1
	GCN4_motif	分生组织特异性激活元件 cis-Regulatory element involved in endosperm expression	4
	MSA-like	参与细胞周期调控的顺式作用元件 cis-Acting element involved in cell cycle regulation	1
	O2-site	玉米醇溶蛋白代谢调控中的顺式调控元件 cis-Acting regulatory element involved in zein metabolism regulation	1

基因ID Gene ID	名称 Name	基因注释 Gene annotation	细胞定位 Cellular localization
PH02Gene46466.t1/PH02Gene44860.t3/ PH02Gene48937.t1	PheCRL	叶绿体，发色团裂解酶CRL Chromophore lyase CRL，chloroplastic	叶绿体，细胞核 Chloroplast，Nucleus
PH02Gene22404.t1	PhePAM16	线粒体导入内膜转位酶亚基 PAM16 like 2 Mitochondrial import inner membrane translocase subunit PAM16 like 2	叶绿体，线粒体 Chloroplast， Mitochondrion
PH02Gene15920.t1	PheR40C1	Ricin B样凝集素R40C1 Ricin B-like lectin R40C1	线粒体Mitochondrion
PH02Gene17378.t1/PH02Gene33021.t2	PheNARS	天冬酰胺-tRNA连接酶 Asparagine-tRNA ligase，	细胞质Cytoplasm
PH02Gene05499.t1	PheTIFY11b	TIFY 11b蛋白 Protein TIFY 11b	细胞核 Nucleus
PH02Gene16416.t1	PheWD	含有WD重复序列的蛋白 WD repeat-containing protein C2A9.03-like	细胞核Nucleus
PH02Gene01990.t1	PheD6PKL1	丝氨酸/苏氨酸蛋白激酶D6PKL1 Serine/threonine-protein kinase D6PKL1	细胞核Nucleus
PH02Gene25370.t1	PheBTH	叶特异性硫蛋白 Leaf-specific thionin	细胞核Nucleus
PH02Gene38247.t1	PheAGUK	三螺旋转录因子ASIL2 Trihelix transcription factor ASIL2	叶绿体Chloroplast
PH02Gene01871.t1	PheAPO4	APO蛋白4 APO protein 4	叶绿体Chloroplast
PH02Gene10760.t1	PheFKBP12	肽基脯氨酰顺反异构酶FKBP12 Peptidyl-prolyl cis-trans isomerase FKBP12	液泡Vacuole
PH02Gene46313.t2	PheF3HL	2-氧戊二酸依赖性双加氧酶 Probable 2-oxoglutarate-dependent dioxygenase	细胞质Cytoplasm
PH02Gene20388.t1	PhePVAP13	EGG APPARATUS-1蛋白 Protein EGG APPARATUS-1	细胞膜，高尔基体 Cell membrane， Golgi apparatus
PH02Gene34419.t1/PH02Gene07485.t1	PheGluD-1	12S种子贮藏球蛋白112S seed storage globulin 1	液泡 Vacuole
PH02Gene32854.t1	PheWD40	具有WDR重复结构域的Dynein组装因子1 Dynein assembly factor with WDR repeat domains 1	细胞核Nucleus
PH02Gene16771.t1	PheATPO	ATP合成酶O亚基 ATP synthase subunit O	线粒体 Mitochondrion
PH02Gene03817.t1	PhePLATZ	PLATZ转录因子 PLATZ transcription factor	叶绿体，细胞核 Chloroplast，Nucleus
PH02Gene01385.t1/ PH02Gene04280.t1	PhePSI-H	光系统I反应中心亚基VI Photosystem I reaction centre subunit VI	叶绿体Chloroplast
PH02Gene44231.t1	Phemac	木瓜蛋白酶家族半胱氨酸蛋白酶 Papain family cysteine protease	液泡Vacuole
PH02Gene18928.t1/ PH02Gene17486.t2	PheXOAT3	ESKIMO 1蛋白 GDSL/SGNH-like Acyl- Protein ESKIMO 1	叶绿体Chloroplast
PH02Gene16891.t1	PheDnaJ	候选钙结合蛋白CML23 Putative calcium-binding protein CML23	线粒体Mitochondrion
PH02Gene03137.t1	PheADF7	肌动蛋白解聚因子2 Actin-depolymerizing factor 2	细胞质 Cytoplasm
PH02Gene28214.t1	PheBTH	硫蛋白BTH7 Thionin BTH7	细胞核 Nucleus
PH02Gene16883.t1	PheS1FA	DNA结合蛋白S1FA DNA binding protein S1FA	细胞核 Nucleus
PH02Gene06848.t1	PheSPC4	阳离子过氧化物酶SPC4 Cationic peroxidase SPC4	胞外 Extracellular
PH02Gene23721.t1	PheLRR	候选抗病蛋白 Putative disease resistance protein	胞外 Extracellular
PH02Gene17499.t1	Phe MIF	巨噬细胞迁移抑制因子同源异构体X2 Macrophage migration inhibitory factor homolog isoform X2	胞外 Extracellular
PH02Gene27732.t1		未知蛋白 Uncharacterized protein	胞外 Extracellular
PH02Gene38290.t1	PhePK	丙酮酸激酶 Pyruvate kinase	细胞质 Cytoplasm
PH02Gene46176.t1	PheMYB	转录因子MYB16 Myb-like DNA-binding domain	胞外 Extracellular