猴樟<em>CbADH</em>基因上游调控蛋白筛选与验证

doi:10.3969/j.issn.1674-7968.2026.03.018

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摘要呼吸代谢与植物耐受盐碱胁迫密切相关,乙醇脱氢酶(alcohol dehydrogenase, ADH)是植物进行无氧呼吸的关键酶,在盐碱胁迫下上调表达,对植物耐受盐碱胁迫起到了重要作用。为揭示CbADH基因参与猴樟(Cinnamomum bodinieri)耐受盐碱胁迫的分子机制,本研究以耐碱猴樟品系为材料,克隆CbADH基因启动子序列,通过酵母单杂交技术筛选出CbADH基因上游调控因子,采用双荧光素报告基因技术验证上游调控蛋白对CbADH基因启动子的功能。结果显示,获得的CbADH基因启动子片段长度为1 996 bp;CbADH基因启动子片段中包含光响应调控元件、脱落酸(abscisic acid, ABA)响应元件、分生组织表达响应元件、茉莉酸甲酯(methyl jasmonic acid, MeJA)响应调控元件、GA响应元件、压力响应元件、水杨酸(salicylic acid, SA)响应元件、愈伤组织响应元件、转录因子MYB结合位点、转录因子bHLH结合位点、转录因子WRKY结合位点。通过酵母单杂交技术共获得17个成功注释的上游调控因子,主要包括蛋白激酶超家族、氨基酸转运体、脱落酸调节休眠的中间体1 (mediator of ABA-regulated dormancy 1, MARDⅠ)锌指结构蛋白、拟南芥同源域-亮氨酸拉链(Arabidopsis thaliana homeobox 6, ATHB-6)蛋白、三糖磷酸异构酶蛋白、胚胎发育晚期丰富蛋白(late embryogenesis abundant protein, LEA)、酪氨酸/多巴胺脱羧酶、甘露醇脱氢酶蛋白、ATP合酶和细胞色素B5家族蛋白。双荧光素酶报告显示,CbATHB6蛋白在植物体内正向激活了CbADH基因启动子活性。以上结果表明,转录因子CbATHB6能够与CbADH基因启动子序列结合,通过调控CbADH基因的表达响应盐碱胁迫。本研究对解析猴樟呼吸代谢响应盐碱胁迫的分子机制提供依据。

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关键词 ：乙醇脱氢酶(ADH)基因, 上游调控蛋白筛选与验证, 酵母单杂交, 双荧光素酶报告基因技术, 猴樟

Abstract：Respiratory metabolism is closely related to plant tolerance to saline and alkali stress, and ethanol dehydrogenase is a key enzyme for anaerobic respiration in plants, which is up-regulated under saline and alkali stress and plays an important role in plant tolerance to saline and alkali stress. In order to clarify the molecular mechanism of CbADH gene involved in the salinity stress tolerance of Cinnamomum bodinieri, in this study, the promoter sequence of CbADH gene was cloned using the alkali-tolerant Cinnamomum bodinieri line as the material, and the upstream regulatory factors of CbADH gene was screened out by yeast one-hybridization, and the function of the upstream regulatory proteins on the promoter of CbADH gene was verified by the dual luciferase reporter gene technique. The results showed that the length of the obtained CbADH gene promoter fragment was 1 996 bp; the CbADH gene promoter fragment contained light-responsive regulatory elements, ABA-responsive elements, phloem-expression-responsive elements, MeJA-responsive elements, GA-responsive elements, stress-responsive elements, SA response element, healing tissue response element, transcription factor MYB binding site, transcription factor bHLH binding site, and transcription factor WRKY binding site. A total of 17 successfully annotated reciprocal proteins were obtained by yeast one-hybrid technique, mainly including protein kinase superfamily, amino acid transporters, mediator of ABA-regulated dormancy 1 (MARDⅠ) zinc finger structural proteins, Arabidopsis thaliana homeobox 6 (ATHB-6) proteins, trisaccharide phosphoribosylceramide isomerase proteins, late embryogenesis abundant protein (LEA) proteins, tyrosine/dopamine decarboxylases, mannitol dehydrogenase proteins, ATP synthases, and cytochrome B5 family proteins. According to the results of the dual luciferase reporter gene experiment technology, it was shown that the CbATHB6 protein activated the promoter activity of CbADH gene in plants. Based on these findings, the transcription factor CbATHB6 could bind to the promoter sequence of the CbADH gene and respond to salt-alk stress by regulating the expression of the CbADH gene. This study provides a basis for analyzing the molecular mechanism of respiratory metabolism of Cinnamomum bodinieri in response to saline and alkaline stress.

Key words： Alcohol dehydrogenase (ADH) gene Screening and validation of upstream regulatory protein Yeast one-hybrid Dual-luciferase reporter gene assay Cinnamomum bodinieri

收稿日期: 2025-03-25

中图分类号： S792.23

基金资助:江苏省自然科学基金(BK20231362); 宿迁市自然科学基金(K202316)

通讯作者: ^*21011@squ.edu.cn

引用本文:

张楠, 韩浩章, 张丽华, 赵荣, 李素华, 王芳. 猴樟CbADH基因上游调控蛋白筛选与验证[J]. 农业生物技术学报, 2026, 34(3): 672-684.
ZHANG Nan, HAN Hao-Zhang, ZHANG Li-Hua, ZHAO Rong, LI Su-Hua, WANG Fang. Screening and Validation of Upstream Regulatory Proteins of Cinnamomum bodinieri CbADH Gene. 农业生物技术学报, 2026, 34(3): 672-684.

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https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2026.03.018 或 https://journal05.magtech.org.cn/Jwk_ny/CN/Y2026/V34/I3/672

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