【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。更多还原

【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 Ca ATG8c 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 Ca IAA8 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.更多还原