辣椒CaHsfA2上游转录因子的筛选及耐热功能分析

doi:10.3864/j.issn.0578-1752.2022.16.011

摘要/Abstract

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出现日渐频繁,热胁迫已经成为影响辣椒生产的主要环境因素之一,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技术分析热胁迫下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及CaBES1沉默辣椒植株中CaHsfA2的表达量发现,转录因子CaBES1对CaHsfA2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可以响应热信号,为下一步对其耐热功能研究提供了支撑。辣椒CaBES1被沉默后,通过对比分析沉默植株和对照植株的表型、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表达而抑制辣椒的耐热性。

关键词: 辣椒, CaHsfA2, CaBES1, 耐热性

Abstract:

【Background】 Pepper is widely cultivated as a vegetable around the world. With the increasing frequency of extreme high temperature weather in recent years, the 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 to develop pepper varieties with heat tolerance. 【Objective】 Since the heat shock transcription factor HsfA2 plays important roles in plant heat tolerance, the upstream transcription factors of pepper CaHsfA2 were screened and functionally analyzed in heat-tolerance in this study, in order to provide the theoretical basis for further understanding the mechanisms of heat tolerance of pepper. 【Method】 The 955 bp 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 interactions 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, their dynamic expression under heat stress in pepper heat-tolerant line R9 were analyzed by qRT-PCR technology; the subcellular localization were fulfilled through the transient gene expression technology, and its functional analysis in heat tolerance were performed by using the virus-induced gene silencing technology (VIGS). 【Result】 CaBES1 was identified as the candidate upstream transcription factor of CaHsfA2, and their interactions were confirmed. By the analysis of Dual-Luciferase system and CaHsfA2 expression in CaBES1-silenced pepper plants, it was suggested that CaBES1 negatively regulated 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 performed its biological functions. By dynamic expression pattern analysis, under heat stress, the expression level of CaBES1 decreased firstly and then increased, which also indicated that CaBES1 could respond to heat signal and laid 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. 【Conclusion】 CaBES1 inhibited pepper heat tolerance by negatively regulating the expression of CaHsfA2.

Key words: pepper, CaHsfA2, CaBES1, heat stress

刘瑞瑶,黄国弘,李海艳,梁敏敏,逯明辉. 辣椒CaHsfA2上游转录因子的筛选及耐热功能分析[J]. 中国农业科学, 2022, 55(16): 3200-3209.

LIU RuiYao,HUANG GuoHong,LI HaiYan,LIANG MinMin,LU MingHui. Screening and Functional Analysis in Heat-Tolerance of the Upstream Transcription Factors of Pepper CaHsfA2[J]. Scientia Agricultura Sinica, 2022, 55(16): 3200-3209.

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用途 Application	名称 Name	序列 Sequence
酵母单杂交 Y1H	T7	TAATACGACTCACTATAGG
	3AD	GAGATGGTGCACGATGCACAGT
	pCaHsfA2-F	CGAGCTCTAAAAGCAAATGTCTGAAACGAGT
	pCaHsfA2-R	GCGTCGACTATCTTTTTTCTTCTTCAGTCGCT
	AD-CaBES1-F	GCCATGGAGGCCAGTGAATTCATGACATCGGGAACAAGG
	AD-CaBES1-R	CAGCTCGAGCTCGATGGATCCTCTTGTCTTAGAACTCCCAA
互作关系验证 Verification of interactive relationship	LUC-pHsfA2-F	GCGTCGACTAAAAGCAAATGTCTGAAACGAGT
	LUC-pHsfA2-R	CATGCCATGGTATCTTTTTTCTTCTTCAGTCGCT
	SK-BES1-F	GCTCTAGAATGACATCGGGAACAAGGA
	SK-BES1-R	GGAATTCTCTTGTCTTAGAACTCCCA
亚细胞定位 Subcellular localization	pART27-BES1-F	GATGAACTATACAAAGAATTCATGACATCGGGAACAAGG
亚细胞定位 Subcellular localization	pART27-BES1-R	TTCAGGCCTCCCGGGGGTACCTCTTGTCTTAGAACTCCCAA
qRT-PCR	qCaBES1-F	TTCGCTACCCGTTCTTTC
	qCaBES1-R	GTGATAGACCCTCCATTTTG
	qCaHsfA2-F	GTAGCA TCAGTAGCCACAGC
	qCaHsfA2-R	CAAGCAACTCTTCCCAAATA

元件 Element	序列 Sequence	位置 Position	数量 Amount	功能 Function
ABRE	ACGTG	61(-)	1	参与脱落酸信号转导途径 Involved in the signal transduction pathway of ABA
HSE	GAANNTTC	36(+), 437(+) 725(+), 822(+)	4	响应热胁迫 Responding to heat stress
E-box	CANNTG	7(+), 465(+) 716(+), 805(+)	4	BES1和BZR1结合位点 BES1 and BZR1 binding site
ARE	AAACCA	53(-) 899(+) 236(-)	3	厌氧诱导必需元件 Necessary for anaerobic induction
CGTCA/TGACG-motif	CGTCA/ TGACG	475(±)	1	参与茉莉酸响应 Involved in jasmonic acid reponsiveness
TCA-element	CCATCTTTTT	804(+)	1	参与水杨酸反应 Involved in salicylic acid reponsiveness