辣椒MADS-box转录因子基因CaAGL61的耐热功能分析

doi:10.3864/j.issn.0578-1752.2025.08.011

摘要/Abstract

摘要：

【目的】MADS-box转录因子是植物最大的转录因子家族之一，在植物生长发育和胁迫响应中均发挥重要功能。前期通过转录组数据获得一个辣椒热响应基因，该基因编码MADS-box转录因子家族成员CaAGL61（Agamous-like MADS-box protein AGL61）。在此基础上，进一步研究CaAGL61在辣椒耐热性调控中的功能，为深入了解辣椒耐热分子机制提供理论参考，为辣椒耐热性的遗传改良提供基因位点。【方法】通过SMART在线工具预测CaAGL61保守结构域，使用MEGA7构建辣椒和其他植物物种AGL61蛋白的系统发育树，利用实时荧光定量PCR技术探究CaAGL61在辣椒中的表达模式，运用烟草亚细胞定位技术和酵母双杂交自激活系统检测CaAGL61的转录因子特性，利用病毒诱导的基因沉默技术和基因瞬时过表达技术探究CaAGL61表达对辣椒耐热性的影响。【结果】CaAGL61编码179个氨基酸，包含一个MADS结构域，在系统进化方面高度保守。CaAGL61在辣椒花器官中的表达量最高、其次是茎和果实，根中的表达量最低；进一步分析发现CaAGL61的表达量随着花器官的成熟而增加，尤其在授粉坐果期的花药中表达量最高；45 ℃高温处理显著上调了CaAGL61的表达水平。亚细胞定位显示，CaAGL61定位于细胞核中；酵母转录激活分析表明，CaAGL61具有转录激活活性。CaAGL61沉默植株的耐热性显著增强，热胁迫处理后，与对照相比，CaAGL61沉默植株生长点萎蔫程度减轻，叶片相对电导率降低，丙二醛含量、死细胞和活性氧积累减少，而叶绿素含量升高。相反，CaAGL61瞬时过表达降低了辣椒的耐热性，与对照相比，表现为植株受热胁迫损伤程度更严重，叶片相对电导率升高，丙二醛含量、死细胞和活性氧积累增多，叶绿素含量下降。【结论】鉴定了一个辣椒热响应MADS-box转录因子基因CaAGL61，该基因通过加剧氧化胁迫而负调控辣椒耐热性。

关键词: 辣椒, 耐热, MADS-box转录因子, CaAGL61, 负调控

Abstract:

【Objective】 MADS-box transcription factors are one of the largest transcription factor families in plants, and play important roles in plant growth, development, and stress responses. Previously, based on transcriptome data of pepper, a heat-responsive gene, Agamous-like MADS-box protein61 (AGL61), was identified as a MADS-box transcription factor. However, the function of CaAGL61 in pepper heat stress remains unknown. In this study, we explored the molecular function of CaAGL61 in heat tolerance of pepper plants, providing the regulatory insights of CaAGL61 as a potential locus for genetic improvement. 【Method】 The SMART online tool was used to predict the conserved domain of CaAGL61, and a phylogenetic tree of AGL61 in pepper and other plant species was constructed using MEGA7. The expression pattern of CaAGL61 was analyzed by quantitative real-time PCR. The subcellular localization of CaAGL61 was examined in tobacco, and its transcriptional regulatory role was investigated using a yeast two-hybrid system (Y2H). The effects of CaAGL61 on heat tolerance of pepper were accessed using transgenic plants generated by virus-induced gene silencing (VIGS) and transient overexpression techniques.【Result】CaAGL61 encodes a protein of 179 amino acids, containing a MADS domain and exhibiting high evolutionary conservation. CaAGL61’s expressional level was relatively high in the flower of pepper, followed by the stem and fruit, and lowest in the root. Further analysis reveals that the expression of CaAGL61 increased along with flower maturation process, peaking in the anther during pollination and fruit setting. High temperature treatment at 45 ℃ significantly upregulates the expression of CaAGL61. Subcellular localization showed that CaAGL61 is located in the nucleus, and yeast transcriptional activation assays indicates that CaAGL61 has transcriptional activation activity. Silencing of CaAGL61 significantly enhances heat tolerance of pepper. Comparing with control, CaAGL61-silenced plants exhibit reduced wilting in plant growth points, lower relative electrolyte leakage, reduced content of malondialdehyde, and accumulation of dead cells and reactive oxygen species (ROS), and increased content of chlorophyll under heat stress. However, transient overexpression of CaAGL61 reduces the heat tolerance of pepper, indicated by more severe heat-stress damage, higher relative electrolyte leakage, increased content of malondialdehyde, and accumulation of dead cells and ROS, and decreased content of chlorophyll compared to control. 【Conclusion】A heat-responsive MADS-box transcription factor gene, CaAGL61, was identified in pepper. This gene negatively regulates pepper heat tolerance by exacerbating oxidative stress.

Key words: pepper, heat tolerance, MADS-box transcription factor, CaAGL61, negative regulation

王梦园, 魏倩睿, 李海艳, 杨巧敏, 宇军, 黄炜, 逯明辉. 辣椒MADS-box转录因子基因CaAGL61的耐热功能分析[J]. 中国农业科学, 2025, 58(8): 1604-1616.

WANG MengYuan, WEI QianRui, LI HaiYan, YANG QiaoMin, YU Jun, HUANG Wei, LU MingHui. Functional Analysis of MADS-box Transcription Factor Gene CaAGL61 in Heat Tolerance of Pepper[J]. Scientia Agricultura Sinica, 2025, 58(8): 1604-1616.

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用途 Purpose	名称 Name	序列 Sequence (5′-3′)
亚细胞定位及基因过表达 Subcellular localization and gene overexpression	pART27-GFP-CaAGL61-F	GATGAACTATACAAAGAATTCATGCAAAATCTACTTGTTGAAG
	pART27-GFP-CaAGL61-R	CAGGACTCTAGATTAGGTACCCTAAGCAAACAAAGCTTGAAAA
基因沉默 Gene-silencing	TRV2:CaAGL61-F	GCTCTAGAGTTCTCGTGTTGCCTCTATTG
基因沉默 Gene-silencing	TRV2:CaAGL61-R	CGGGATCCGATGGTTCCGTGCATCTACTA
转录激活活性分析 Activity analysis of transcriptional activation	BD-CaAGL61-F	ATGGCCATGGAGGCCGAATTCATGCAAAATCTACTTGTTGAAG
转录激活活性分析 Activity analysis of transcriptional activation	BD-CaAGL61-R	CCGCTGCAGGTCGACGGATCCCTAAGCAAACAAAGCTTGAAAAG
基因表达分析 Analysis of gene expression	qCaAGL61-F	GGCCTTATACCTGCGGAGAC
	qCaAGL61-R	GTCGAAGACCATCTCGACCC
	qCaUbi3-F	TGTCCATCTGCTCTCTGTTG
	qCaUbi3-R	CACCCCAAGCACAATAAGAC
	qCaHsfA2-F	GTAGCATCAGTAGCCACAGC
	qCaHsfA2-R	CAAGCAACTCTTCCCAAATA
	qCaHsfA3-F	CGAAAGTATGATGAAAGAAGAGG
	qCaHsfA3-R	ATAGTTGCCAAGACCACCC
	qCaHsp70.1-F	CAGGTGTGCTAGTTCAGGTGT
	qCaHsp70.1-R	TGACCTGAGGCACTCCTCTT