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

Abstract

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

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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References 35

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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

Screening and Functional Analysis in Heat-Tolerance of the Upstream Transcription Factors of Pepper CaHsfA2

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