Main conclusionHSP60 gene family in pepper was analyzed through bioinformatics along with transcriptional regulation against multiple abiotic and hormonal stresses. Furthermore, the knockdown of CaHSP60-6 increased sensitivity to heat stress.AbstractThe 60 kDa heat shock protein (HSP60) also known as chaperonin (cpn60) is encoded by multi-gene family that plays an important role in plant growth, development and in stress response as a molecular chaperone. However, little is known about the HSP60 gene family in pepper (Capsicum annuum L.). In this study, 16 putative pepper HSP60 genes were identified through bioinformatic tools. The phylogenetic tree revealed that eight of the pepper HSP60 genes (50%) clustered into group I, three (19%) into group II, and five (31%) into group III. Twelve (75%) CaHSP60 genes have more than 10 introns, while only a single gene contained no introns. Chromosomal mapping revealed that the tandem and segmental duplication events occurred in the process of evolution. Gene ontology enrichment analysis predicted that CaHSP60 genes were responsible for protein folding and refolding in an ATP-dependent manner in response to various stresses in the biological processes category. Multiple stress-related cis-regulatory elements were found in the promoter region of these CaHSP60 genes, which indicated that these genes were regulated in response to multiple stresses. Tissue-specific expression was studied under normal conditions and induced under 2 h of heat stress measured by RNA-Seq data and qRT-PCR in different tissues (roots, stems, leaves, and flowers). The data implied that HSP60 genes play a crucial role in pepper growth, development, and stress responses. Fifteen (93%) CaHSP60 genes were induced in both, thermo-sensitive B6 and thermo-tolerant R9 lines under heat treatment. The relative expression of nine representative CaHSP60 genes in response to other abiotic stresses (cold, NaCl, and mannitol) and hormonal applications [ABA, methyl jasmonate (MeJA), and salicylic acid (SA)] was also evaluated. Knockdown of CaHSP60-6 increased the sensitivity to heat shock treatment as documented by a higher relative electrolyte leakage, lipid peroxidation, and reactive oxygen species accumulation in silenced pepper plants along with a substantial lower chlorophyll content and antioxidant enzyme activity. These results suggested that HSP60 might act as a positive regulator in pepper defense against heat and other abiotic stresses. Our results provide a basis for further functional analysis of HSP60 genes in pepper.

中文翻译：

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敲除 CaHSP60-6 可增强辣椒 (Capsicum annuum L.) 对热应激的敏感性

主要结论通过生物信息学分析辣椒中的HSP60基因家族以及针对多种非生物和激素胁迫的转录调控。此外，CaHSP60-6 的敲低增加了对热应激的敏感性。 摘要 60 kDa 热休克蛋白 (HSP60) 也称为伴侣蛋白 (cpn60)，由在植物生长、发育和胁迫中起重要作用的多基因家族编码。作为分子伴侣的反应。然而，人们对辣椒（Capsicum annuum L.）中的 HSP60 基因家族知之甚少。在这项研究中，通过生物信息学工具鉴定了 16 个推测的辣椒 HSP60 基因。系统发育树显示，辣椒 HSP60 基因中有 8 个（50%）聚集在 I 组中，3 个（19%）聚集在 II 组中，5 个（31%）聚集在 III 组中。十二个 (75%) CaHSP60 基因有超过 10 个内含子，而只有一个基因不包含内含子。染色体作图显示，进化过程中发生了串联和节段重复事​​件。基因本体富集分析预测，CaHSP60 基因以 ATP 依赖性方式负责蛋白质折叠和再折叠，以响应生物过程类别中的各种压力。在这些 CaHSP60 基因的启动子区域中发现了多个与应激相关的顺式调节元件，这表明这些基因响应于多种应激而受到调节。在正常条件下研究组织特异性表达，并在不同组织（根、茎、叶和花）中通过 RNA-Seq 数据和 qRT-PCR 测量在 2 小时的热应激下诱导。数据暗示 HSP60 基因在辣椒的生长、发育、和压力反应。15 个 (93%) CaHSP60 基因在热处理下的热敏 B6 和耐热 R9 系中均被诱导。还评估了九个代表性 CaHSP60 基因响应其他非生物胁迫（冷、NaCl 和甘露醇）和激素应用 [ABA、茉莉酸甲酯 (MeJA) 和水杨酸 (SA)] 的相对表达。CaHSP60-6 的敲除增加了对热休克处理的敏感性，这可以通过沉默辣椒植物中更高的相对电解质泄漏、脂质过氧化和活性氧积累以及显着降低的叶绿素含量和抗氧化酶活性来证明。这些结果表明，HSP60 可能作为辣椒防御热和其他非生物胁迫的正调节剂。