中国农业科学

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最新录用:辣椒CaHsfA2上游转录因子的筛选及耐热功能分析

刘瑞瑶,黄国弘,李海艳,梁敏敏,逯明辉   

  1. 西北农林科技大学园艺学院,陕西杨凌 712100
  • 出版日期:2022-06-24 发布日期:2022-06-24

Screening and Functional analysis in heat-tolerance of the upstream transcription factors of pepper CaHsfA2

LIU RuiYao, HUANG GuoHong, LI HaiYa1, LIANG MinMin, LU MingHui   

  1. College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi
  • Published:2022-06-24 Online:2022-06-24

摘要: 【背景】辣椒作为一种在世界范围内普遍栽培的蔬菜,具有喜温不耐热的特性。随着近些年极端高温天气日渐频繁的出现,热胁迫已经成为影响辣椒生产的主要环境因素之一,因此明确辣椒的耐热机制进而培育耐热品种对辣椒生产具有重要的意义。【目的】热激转录因子HsfA2在植物耐热性中发挥重要作用,筛选辣椒CaHsfA2上游的转录因子,并分析后者在辣椒耐热性形成中的作用,为进一步阐明辣椒耐热分子机制提供理论依据。【方法】以CaHsfA2起始密码子上游的955 bp启动子序列为诱饵,利用酵母单杂交(Y1H)技术,筛选CaHsfA2的上游转录因子,并通过Y1H点对点杂交、双荧光素酶报告系统(Dual-Luciferase)与萤火素酶互补技术(LCA)进一步验证二者之间的互作关系。利用qRT-PCR技术分析胁迫下CaHsfA2上游转录因子在辣椒耐热品系R9中的动态表达模式;利用基因瞬时表达技术分析上游转录因子的亚细胞定位;利用病毒诱导的基因沉默技术(VIGS),分析CaHsfA2上游转录因子的耐热功能。【结果】筛选获得了CaHsfA2上游转录因子CaBES1,验证了二者的互作关系,通过分析双荧光素酶报告系统的结果、CaBES1沉默辣椒植株中CaHsfA2的表达量发现,转录因子CaBES1CaHsfA2具有转录抑制作用。亚细胞定位结果表明,CaBES1在细胞膜和细胞核上均有表达,热胁迫处理后细胞核内的荧光信号增强,符合其发挥生物学功能时由细胞质向细胞核转移的特性;动态表达模式分析表明,热胁迫下,CaBES1表达水平呈现先降低后升高的变化趋势,这也说明CaBES1是可以响应热信号的,为下一步对它的耐热功能研究提供了支撑。辣椒CaBES1基因被沉默后,通过对比分析沉默植株和对照植株在表型、相对电导率、叶绿素含量等指标发现,CaBES1的沉默表达提高了CaHsfA2表达量并增强了辣椒的耐热性。【结论】CaBES1通过负调控CaHsfA2表达从而抑制辣椒的耐热性。


关键词: 辣椒, CaHsfA2, CaBES1, 耐热性

Abstract: BackgroundPepper is widely cultivated as a vegetable around the world. With the increasing frequency of extreme high temperature weather in recent years, heat stress has become one of the main environmental factors affecting pepper productivity, due to its feature of warm-prone but heat-sensitive. Therefore, it is very important for pepper production to clarify its mechanisms supporting heat-tolerance and then develop pepper varieties with heat tolerance. ObjectiveSince the heat shock transcription factor HsfA2 plays important roles in plant heat tolerance, the upstream transcription factor of pepper CaHsfA2 was screened and functionally analyzed in heat-tolerance in this study, in order to provide theoretical basis for further understanding the mechanisms of heat tolerance of pepper.MethodThe 955bp promoter sequence upstream of start codon of CaHsfA2 was used as the bait, the yeast one-hybrid (Y1H) technology was applied to screen the upstream transcription factors of CaHsfA2, and their interaction were checked by Y1H point-to-point hybridization, dual-luciferase reporter system (Dual-Luciferase), and LUC assay (LCA). For the candidate upstream transcription factors of CaHsfA2, its dynamic expression under heat stress in pepper heat-tolerant line R9 were analyzed by qRT-PCR technology; its subcellular localization were fulfilled through the transient gene expression technology, and its functional analysis in heat tolerance were performed using the virus-induced gene silencing technology (VIGS). ResultsCaBES1 was identified as the candidate upstream transcription factor of CaHsfA2, and their interaction was confirmed. By the analysis of Dual-Luciferase system and CaHsfA2 expression in CaBES1-silenced pepper plants, it is suggested that CaBES1 negatively regulates the transcription of CaHsfA2. The result of subcellular localization showed that CaBES1 was expressed in both cell membrane and nucleus. After heat stress treatment, the fluorescence signal in the nucleus was enhanced, which was consistent with the property of CaBES1 transferring from cytoplasm to nucleus when it performs its biological functions. By dynamic expression pattern analysis, under heat stress, the expression level of CaBES1 decreased firstly and then increased, which also indicates that CaBES1 can respond to heat signal and lays a foundation for the further functional study in heat tolerance. After CaBES1 was silenced in pepper, by comparing the phenotype, relative electrical conductivity and chlorophyll content of silenced plants and control plants, it was inferred that silencing of CaBES1 increased the expression of CaHsfA2 and enhanced the heat tolerance of pepper. ConclusionCaBES1 inhibits pepper heat tolerance by negatively regulating the expression of CaHsfA2.

Key words: pepper, CaHsfA2, CaBES1, heat stress