辣椒自噬相关蛋白CaATG8c互作蛋白CaIAA8的耐热功能分析

doi:10.3864/j.issn.0578-1752.2025.22.013

摘要/Abstract

摘要：

【目的】自噬是真核细胞中一种保守的蛋白质降解系统，在植物抗逆中发挥重要作用。自噬相关基因CaATG8c沉默表达会降低辣椒耐热性，通过酵母双杂交技术筛选获得一个CaATG8c的候选互作蛋白——生长素响应蛋白CaIAA8。分析CaIAA8的序列特征，验证其与CaATG8c的互作关系，并研究其对辣椒耐热性的调控作用，为阐明辣椒耐热机制及选育辣椒耐热品种提供参考。【方法】使用NCBI数据库的BLASTP工具、IQ-TREE软件、MEME在线工具分别进行CaIAA8蛋白序列比对、系统发育树构建和保守基序分析，使用ExPASy、SignalP-6.0、TMHMM-2.0、NetPhos工具分别进行蛋白理化性质分析，以及信号肽、跨膜域和磷酸化位点预测，利用在线工具SOPMA和SWISS-MODEL进行蛋白二、三级结构预测。利用酵母双杂交和荧光素酶互补试验验证CaATG8c和CaIAA8的相互作用，采用基因瞬时表达技术探究CaIAA8的亚细胞定位，利用实时荧光定量PCR技术分析CaIAA8在辣椒中的表达模式，使用病毒诱导的基因沉默技术和基因瞬时过表达技术研究其对辣椒耐热性的影响。【结果】CaIAA8编码349个氨基酸，蛋白相对分子量为37.48 kDa，等电点为7.52，具有65个预测的磷酸化位点。CaIAA8为碱性亲水性蛋白，结构不稳定。CaIAA8主要定位于细胞质，且与叶绿体存在共定位。CaIAA8在辣椒茎中表达量最高，其次是果和花，叶中的表达量较低。45 ℃热胁迫处理期间，CaIAA8表达水平呈下降趋势。与对照植株相比，CaIAA8沉默辣椒植株的耐热性增强，表现为热胁迫条件下叶片萎蔫程度减轻、相对电导率较低、死细胞和H₂O₂积累较少，并且热激蛋白基因和热激转录因子基因表达量增高。相反，与对照相比，CaIAA8瞬时过表达降低了辣椒的耐热性，表现为植株热损伤程度更严重，死细胞和H₂O₂积累增多。【结论】验证了CaIAA8与CaATG8c的相互作用，且CaIAA8负调控辣椒耐热性。

关键词: 辣椒, 耐热, 自噬, CaATG8c, 生长素响应蛋白, CaIAA8

Abstract:

【Objective】Autophagy is a conserved protein degradation system in eukaryotic cells and plays an important role in plant stress tolerance. Previous studies showed that silencing the autophagy-related gene CaATG8c reduces heat tolerance in pepper. A candidate interacting protein of CaATG8c, the auxin responsive protein CaIAA8, was identified via yeast two-hybrid screening. This study aimed to analyze the sequence characteristics of CaIAA8, confirm its interaction with CaATG8c, and investigate its role in regulating heat tolerance in pepper, thereby providing insights into the molecular mechanisms of heat tolerance and supporting the breeding of heat-tolerant cultivars. 【Method】 Protein sequence alignment, phylogenetic analysis, and conserved motif identification of CaIAA8 were conducted using BLASTP (NCBI), IQ-TREE, and MEME, respectively. Protein physicochemical properties, signal peptide, transmembrane domain, and phosphorylation sites were predicted using ExPASy, SignalP-6.0, TMHMM-2.0, and NetPhos, respectively. Protein secondary and tertiary structures were predicted using SOPMA and SWISS-MODEL respectively. The interaction between CaATG8c and CaIAA8 was validated by yeast two-hybrid and luciferase complementation assays. Subcellular localization of CaIAA8 was examined by transient expression in tobacco leaves. The expression pattern of CaIAA8 gene in pepper was analyzed using real-time quantitative PCR (RT-qPCR), and its function in heat tolerance was further evaluated by virus-induced gene silencing (VIGS) and transient overexpression in pepper. 【Result】CaIAA8 encodes a 349-amino-acid protein with a molecular weight of 37.48 kDa and an isoelectric point of 7.52. It contains 65 predicted phosphorylation sites and is a basic, hydrophilic, and structurally unstable protein. CaIAA8 localizes primarily to the cytoplasm and co-localizes with chloroplasts. Its expression is highest in stems, followed by fruits and flowers, and lowest in leaves. Under 45 ℃ heat stress, CaIAA8 transcript level decreases. Silencing CaIAA8 enhances pepper heat tolerance, as evidenced by reduced leaf wilting, lower relative electrolyte leakage, decreased cell death and H₂O₂ accumulation, and increased expression levels of heat shock protein and heat shock transcription factor genes. In contrast, transient overexpression of CaIAA8 reduces pepper heat tolerance, leading to more severe heat-induced damage and elevated levels of cell death and H₂O₂. 【Conclusion】CaIAA8 physically interacts with CaATG8c and functions as a negative regulator of heat tolerance in pepper.

Key words: pepper, heat tolerance, autophagy, CaATG8c, auxin responsive protein, CaIAA8

李倪菲, 杨巧敏, 杨可成, 邢誉腾, 王梦园, 臧天宝, 逯明辉. 辣椒自噬相关蛋白CaATG8c互作蛋白CaIAA8的耐热功能分析[J]. 中国农业科学, 2025, 58(22): 4732-4745.

LI NiFei, YANG QiaoMin, YANG KeCheng, XING YuTeng, WANG MengYuan, ZANG TianBao, LU MingHui. Functional Analysis of CaIAA8, An Interacting Protein of the Autophagy-Related Protein CaATG8c, in the Heat Tolerance of Pepper[J]. Scientia Agricultura Sinica, 2025, 58(22): 4732-4745.

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用途Purpose	名称Name	序列Sequence (5′-3′)
酵母双杂交 Y2H	T7	TAATACGACTCACTATAGG
	3AD	GAGATGGTGCACGATGCACAGT
	AD-CaIAA8-F	GCCATGGAGGCCAGTGAATTC
	AD-CaIAA8-R	CAGCTCGAGCTCGATGGATCC
	BD-CaATG8c-F	ATGGCCATGGAGGCCGAATTCATGGCGAAGAGTTCATTC
	BD-CaATG8c-R	CCGCTGCAGGTCGACGGATCCTTACACAAAGTTAAGGTCCC
荧光素酶互补成像 LCI	CLuc-F	GTGTTTGTGGACGAAGTACC
	CLuc-R	CCATTTCACAGTTCGATAGCG
	NLuc-F	TGATGTGATATCTCCACTGACG
	NLuc-R	GGGCGTATCTCTTCATAGCC
	Cluc-CaIAA8-F	TACGCGTCCCGGGGCGGTACC
	Cluc-CaIAA8-R	ACGAAAGCTCTGCAGGTCGAC
	NLuc-CaATG8c-F	ACGAAAGCTCTGCAGGTCGACCTACTGATGCACGGAGGAATC
	NLuc-CaATG8c-R	CGCGTACGAGATCTGGTCGACCACAAAGTTAAGGTCCCCAAAT
亚细胞定位及基因过表达 Subcellular localization and gene overexpression	pART27-CaIAA8-F	GATGAACTATACAAAGAATTCA
	pART27-CaIAA8-R	CAGGACTCTAGATTAGGTACCC
基因表达分析 Analysis of gene expression	CaUbi3-F	TGTCCATCTGCTCTCTGTTG
	CaUbi3-R	CACCCCAAGCACAATAAGAC
	qCaIAA8-F	AAAGGCTCAGGTTGTGGGTT
	qCaIAA8-R	AACATTTCCTTCCCAGGAGC
	qCaHsfA2-F	GTAGCATCAGTAGCCACAGC
	qCaHsfA2-R	CAAGCAACTCTTCCCAAATA
	qCaHSP16.4-F	ATGTCAAAGATGATCAGCTTATTG
	qCaHSP16.4-R	GGGTACTCAACACGGGACAC
	qCaHSP25.9-F	AGGAGACGACGCCTTAGTAG
	qCaHSP25.9-R	TCTGACCCTTCTTTCTCTTCC
	qCaHSP70.1-F	CAGGTGTGCTAGTTCAGGTGT
	qCaHSP70.1-R	TGACCTGAGGCACTCCTCTT
基因沉默 Gene-silencing	TRV2:CaIAA8-F	GCTCTAGAATGTCTCCACCACT
基因沉默 Gene-silencing	TRV2:CaIAA8-R	CGGGATCCCTCTTCAACCTTTGT