茶树‘英红9号’乙胺合成关键酶基因CsAlaDC转录因子的筛选

doi:10.3864/j.issn.0578-1752.2025.12.012

摘要/Abstract

摘要：

【目的】茶氨酸是由底物乙胺和谷氨酸经茶氨酸合成酶催化合成。乙胺是茶树茶氨酸生成的主要限制因子，其由丙氨酸脱羧酶（CsAlaDC）催化丙氨酸脱羧而来。通过酵母单杂交从茶叶文库中筛选获得调控CsAlaDC的转录因子，从转录因子角度探究茶树体内乙胺合成调控的分子机制，为茶氨酸合成和后续开展茶树遗传改良提供理论基础和技术参考。【方法】以‘英红9号’茶树为材料，通过PCR克隆CsAlaDC启动子，以其为诱饵，利用酵母单杂交从文库中筛选获得候选转录因子，克隆候选转录因子的CDS，并回转验证。利用生物信息学方法对候选转录因子进行蛋白结构域、蛋白大小及可溶性分析，借助双荧光素酶试验探究候选转录因子对CsAlaDC的表达调控作用，并用亚细胞定位技术测定候选转录因子在细胞内的分布情况。【结果】克隆获得‘英红9号’茶树CsAlaDC启动子，包含真核生物基本的TATA-box和CAAT-box等启动子核心元件，I-box、G-Box、TCT-motif、TCCC-motif、Box4等光响应相关元件，CAT-box分生组织表达相关顺式作用元件，以及ABRE脱落酸响应元件、TC-rich repeats防御和压力反应顺式作用元件，还有MYB、MYC和WRKY等转录因子家族识别结合位点。以CsAlaDC启动子为诱饵进行酵母单杂交，从‘英红9号’cDNA文库中筛选获得17个结合蛋白，经初筛，获得5个候选转录因子，克隆CDS，并回转验证，获得3个转录因子：CsFBX、CsBBX和CsASR。双荧光素酶试验表明，CsBBX和CsASR对CsAlaDC具有转录激活作用，CsFBX无显著调控活性。亚细胞定位表明，CsFBX和CsBBX定位于细胞核，CsASR定位于细胞核和细胞质。【结论】从‘英红9号’cDNA文库中筛选获得3个调控乙胺合成关键酶基因CsAlaDC的转录因子：CsFBX、CsBBX和CsASR，均能结合CsAlaDC启动子，且CsBBX和CsASR对CsAlaDC具有转录激活作用。

关键词: 茶, 茶氨酸, 乙胺, 丙氨酸脱羧酶, 转录因子, 转录调控

Abstract:

【Objective】Theanine is synthesized from substrates ethylamine and glutamic acid catalyzed by theanine synthase. Ethylamine, the primary limiting factor in theanine biosynthesis in tea plants (Camellia sinensis), is generated via the decarboxylation of alanine catalyzed by alanine decarboxylase (CsAlaDC). This study aims to identify transcription factors regulating CsAlaDC through yeast one-hybrid screening of a tea plant cDNA library, thereby elucidating the molecular mechanisms underlying ethylamine biosynthesis regulation from a transcriptional perspective. The findings provide a theoretical foundation and technical references for advancing theanine biosynthesis and genetic improvement in tea plants.【Method】Using the Yinghong 9 tea cultivar, the promoter of CsAlaDC was cloned via PCR and employed as bait for yeast one-hybrid screening to identify candidate transcription factors from a cDNA library. Coding sequences (CDS) of candidate transcription factors were cloned and reversed to validate them. Bioinformatics analyses were conducted to characterize protein domains, molecular weights, and solubility of the candidates. Dual-luciferase assays were performed to assess transcriptional regulation of CsAlaDC by the candidate transcription factors, while subcellular localization experiments determined their cellular distribution.【Result】The CsAlaDC promoter cloned from the Yinghong 9 tea cultivar contained core eukaryotic promoter elements such as TATA-box and CAAT-box, along with light-responsive motifs (I-box, G-Box, TCT-motif, TCCC-motif, Box4), a meristem-specific CAT-box cis-element, stress-responsive elements (ABRE, TC-rich repeats), and binding sites for transcription factor families including MYB, MYC, and WRKY. Using the CsAlaDC promoter as bait, yeast one-hybrid screening of the Yinghong 9 cDNA library identified 17 potential binding proteins. After preliminary screening, five candidate transcription factors were selected for CDS cloning and reverse validation, ultimately confirming three transcription factors: CsFBX, CsBBX, and CsASR. Dual-luciferase assays demonstrated that CsBBX and CsASR significantly activated CsAlaDC expression, whereas CsFBX exhibited no regulatory effect. Subcellular localization revealed nuclear localization of CsFBX and CsBBX, while CsASR was localized in both the nucleus and cytoplasm.【Conclusion】Three transcription factors (CsFBX, CsBBX, and CsASR) regulating the ethylamine biosynthesis gene CsAlaDC were identified from the Yinghong 9 cDNA library. All three factors bound to the CsAlaDC promoter, with CsBBX and CsASR demonstrating transcriptional activation capabilities.

Key words: Camellia sinensis, theanine, ethylamine, alanine decarboxylase (CsAlaDC), transcription factors (TFs), transcriptional regulation

阮桥君, 毛苗苗, 张媛媛, 林晓蓉, 陈忠正. 茶树‘英红9号’乙胺合成关键酶基因CsAlaDC转录因子的筛选[J]. 中国农业科学, 2025, 58(12): 2427-2438.

RUAN QiaoJun, MAO MiaoMiao, ZHANG YuanYuan, LIN XiaoRong, CHEN ZhongZheng. Screening of Transcription Factors for Key Enzyme Gene CsAlaDC Involved in Ethylamine Synthesis in Yinghong 9 (Camellia sinensis)[J]. Scientia Agricultura Sinica, 2025, 58(12): 2427-2438.

图/表 10

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从文库中筛出与proCsAlaDC互作的候选蛋白"

序号 Sequence number	基因ID Gene ID	功能描述 Function description
1	XM_002269237.5	欧亚葡萄亚硫酸盐氧化酶样蛋白Vitis vinifera sulfite oxidase-like
2	XM_028226709.1	茶树LIMR家族蛋白At5g01460样Camellia sinensis LIMR family protein At5g01460-like
3	XM_028196337.1	茶树锌指蛋白CONSTANS样4类似物Camellia sinensis zinc finger protein CONSTANS-LIKE 4-like
4	XM_028200475.1	茶树果胶酯酶抑制剂3 Camellia sinensis pectinesterase inhibitor 3
5	XM_028252569.1	茶树未表征蛋白Camellia sinensis uncharacterized
6	XM_028216177.1	茶树器官特异性蛋白P4样Camellia sinensis organ-specific protein P4-like
7	XM_028234224.1	茶树60S核糖体蛋白L13-1样Camellia sinensis 60S ribosomal protein L13-1-like
8	XM_028266808.1	茶树可能的核酮糖-5-磷酸异构酶3（叶绿体型） Camellia sinensis probable ribose-5-phosphate isomerase 3, chloroplastic
9	XM_028214394.1	茶树40S核糖体蛋白S3a-2样Camellia sinensis 40S ribosomal protein S3a-2-like
10	KF880380.1	茶树脱落酸胁迫成熟蛋白ASR Camellia sinensis abscisic stress-ripening protein ASR (ASR)
11	XM_028204029.1	茶树F-box蛋白At5g03970样Camellia sinensis F-box protein At5g03970-like
12	XM_034853669.1	河岸葡萄未表征蛋白Vitis riparia uncharacterized
13	NM_001378517.1	欧亚葡萄60S酸性核糖体蛋白P2B样Vitis vinifera 60S acidic ribosomal protein P2B-like
14	XM_028233642.1	茶树可能的半乳糖醛酸转移酶样7 Camellia sinensis probable galacturonosyltransferase-like 7
15	XM_028196820.1	茶树谷氨酸受体2.7样Camellia sinensis glutamate receptor 2.7-like
16	XM_028232263.1	茶树琥珀酸脱氢酶组装因子4（线粒体型样） Camellia sinensis succinate dehydrogenase assembly factor 4, mitochondrial-like
17	XM_010652188.2	欧亚葡萄可能的E3泛素蛋白连接酶XERICO Vitis vinifera probable E3 ubiquitin-protein ligase XERICO

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位点名称 Site name	数量 Quantity	序列 Sequence	功能描述 Function description
CAAT-box	46	CAAAT/CAAT/CAACCAACTCC	启动子和增强子区域常见的顺式作用元件 Common cis-acting element in promoter and enhancer regions
TATA-box	121	TATA	启动子核心序列Core promoter element
I-box	1	AAGATAAGGCT	部分光响应元件Part of a light responsive element
G-Box	2	CACGTT	参与光反应的顺式作用调控元件 Cis-acting regulatory element involved in light responsiveness
TCT-motif	1	TCTTAC	部分光响应元件Part of a light responsive element
TCCC-motif	1	TCTCCCT	部分光响应元件Part of a light responsive element
Box4	6	ATTAAT	部分参与光响应的保守DNA模块 Part of a conserved DNA module involved in light responsiveness
A-box	1	CCGTCC	顺式作用元件Cis-acting regulatory element
CAT-box	1	GCCACT	分生组织表达相关顺式作用元件 Cis-acting regulatory element related to meristem expression
TC-rich repeats	2	ATTCTCTAAC	防御和压力反应的顺式作用元件 Cis-acting element involved in defense and stress responsiveness
ABRE	6	CGTACGTGCA	脱落酸响应元件 Cis-acting element involved in the abscisic acid responsiveness
MYB	1	CAACCA	MYB转录因子识别位点Binding sites for MYB transcription factors
MYC	2	CATTTG	MYC转录因子识别位点Binding sites for MYC transcription factors
ERE	2	ATTTCATA	雌激素反应元件Estrogen response element
W-Box	2	TTGACC	WRKY转录因子识别位点Binding sites for WRKY transcription factors

名称 Name	长度 Length (bp)	分子量 Molecular weight (Da)	氨基酸总数 Number of amino acids	等电点 Theoretical pI	亲疏水性 Hydrophilicity	稳定性 Stability	亚细胞定位 Subcellular localization
CsFBX	1407	54366.06	468	6.73	亲水Hydrophilic	不稳定Unstable	细胞核Nucleus
CsBBX	1080	39446.05	359	5.90	亲水Hydrophilic	不稳定Unstable	细胞核Nucleus
CsASR	546	19889.19	181	5.69	亲水Hydrophilic	稳定Stable	细胞质/细胞核Cytoplasm/Nucleus
CsLIMR	1533	56247.61	510	9.10	疏水Hydrophobic	稳定Stable	质膜Plasma membrane
CsOrgan	375	13840.49	124	4.97	亲水Hydrophilic	稳定Stable	细胞外基质Extracellular matrix