辣椒<i>CaHSP21</i>基因的克隆及表达分析

doi:10.7668/hbnxb.20196169

摘要/Abstract

摘要：

小分子热激蛋白(sHSP)作为分子伴侣在植物抗逆过程中发挥着重要的调控作用。为了研究辣椒中sHSP 的生物学功能,以耐热辣椒材料17CL30为试验材料,对编码sHSP的基因CaHSP21进行克隆和生物信息学分析,并通过实时荧光定量PCR分析其在不同组织和高温胁迫下的表达模式。结果表明,CaHSP21基因编码序列(CDS)全长为705 bp,编码234个氨基酸;CaHSP21理化性质分析结果显示,CaHSP21分子质量和理论等电点分别为25.74 ku和6.53,为中性蛋白。不稳定系数为37.39,是稳定蛋白。CaHSP21含有sHSP家族典型的HSP20结构域,蛋白质二级结构主要由无规则卷曲构成。蛋白质系统进化分析表明,CaHSP21与中国辣椒、下垂辣椒遗传距离最近,且在进化上属于同一分支。组织表达分析结果表明,CaHSP21在辣椒花中表达量最低,果中表达量最高。高温胁迫下的表达模式分析结果表明,CaHSP21受高温胁迫诱导,其相对表达量在高温处理3 h后达到峰值。亚细胞定位结果显示,CaHSP21主要定位于质膜和细胞核中。综上,CaHSP21是一个参与辣椒对高温胁迫响应的关键基因。

关键词: 辣椒, CaHSP21, 高温胁迫, 基因克隆, 表达分析, 生物信息学

Abstract:

Small heat shock proteins(sHSP)play an important regulatory role as molecular chaperones in plant stress resistance.To investigate the biological function of sHSP in pepper,the heat-tolerant pepper material 17CL30 was used as the experimental material.The sHSP-encoding gene CaHSP21 was cloned and subjected to bioinformatics analysis.Its expression patterns in different tissues and under high-temperature stress were analyzed by Quantitative Real-time PCR.The results showed that the full-length CDS of CaHSP21 was 705 bp,encoding 234 amino acids.Physicochemical analysis revealed that the molecular mass and theoretical isoelectric point(pI)of CaHSP21 were 25.74 ku and 6.53,respectively,indicating it is a neutral protein.Its instability index was 37.39,it is classified as a stable protein.CaHSP21 contained the typical HSP20 domain of the sHSP family,and its secondary structure was primarily composed of random coils.Protein phylogenetic analysis showed that CaHSP21 had the closest genetic distance to Capsicum chinense and Capsicum baccatum,and they belonged to the same evolutionary branch.Tissue-specific expression analysis indicated that CaHSP21 expression was lowest in flowers and highest in fruits.Under high-temperature stress,CaHSP21 was induced by high temperature,and its relative expression level peaked after 3 hours of treatment.Subcellular localization results demonstrated that CaHSP21 was localized in the plasma membrane and cell nucleus.In conclusion,CaHSP21 is a key gene involved in pepper's response to high-temperature stress.

Key words: Pepper, CaHSP21, High-temperature stress, Gene cloning, Expression analysis, Bioinformatics

中图分类号:

黄嘉欣, 贾旭, 王田田, 常雪瑞, 王静. 辣椒CaHSP21基因的克隆及表达分析[J]. 华北农学报, 2026, 41(3): 40-47. doi: 10.7668/hbnxb.20196169.

HUANG Jiaxin, JIA Xu, WANG Tiantian, CHANG Xuerui, WANG Jing. Cloning and Expression Analysis of CaHSP21 Gene in Capsicum annuum[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(3): 40-47. doi: 10.7668/hbnxb.20196169.

http://www.hbnxb.net/CN/Y2026/V41/I3/40

图/表 12

表1 试验所用引物

Tab.1 Primers used in the present study

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
CaHSP21-F	ATGGCTTGCACGACTGTTACATG
CaHSP21-R	TTAGTTGACATGCACGTCAATGACC
qCaHSP21-F	ACGTGCAGCATGTCAAAAAC
qCaHSP21-R	GGAATGTCGAATCGCATCTT
Actin-F	CCACCTCTTCACTCTCTGCTCT
Actin-R	ACTAGGAAAAACAGCCCTTGGT
CaHSP21-pART-CAM-EGFP-F	CATTTGGAGAGGACACGCATGGCTTGCACGACTGTTAC
CaHSP21-pART-CAM-EGFP-R	TCGCCCTTGCTCACCATGAAGTTGACATGCACGTCAATGACC

图1 CaHSP21基因的克隆 A.CaHSP21的PCR扩增产物;B.染色体定位;C.基因结构分析。Marker.DNA分子质量标准;1.目的片段。

Fig.1 Cloning of the CaHSP21 A.PCR amplification;B.Chromosome localization;C.Gene structure of CaHSP21 gene.Marker.DNA Marker;1.Target fragment.

图2 CaHSP21全长核苷酸序列及其编码的氨基酸序列

Fig.2 Full-length nucleotide sequence and amino acid sequence of CaHSP21

图3 CaHSP21蛋白保守结构域分析

Fig.3 Conservative domain analysis of CaHSP21 protein

图4 CaHSP21的生物信息学分析 A.跨膜结构;B.信号肽;C.磷酸化位点;D.亲/疏水性分析。

Fig.4 Bioinformatics analysis of CaHSP21 A.Transmembrane structure;B.Signal peptide;C.Phosphorylation potential;D.Affinity/hydrophobicity.

图5 CaHSP21蛋白二级结构预测

Fig.5 Secondary structure prediction of CaHSP21 protein

图6 CaHSP21蛋白三级结构预测

Fig.6 Tertiary structure prediction of CaHSP21 protein

图7 CaHSP21蛋白与同源蛋白的序列比对

Fig.7 Sequence alignment of CaHSP21 protein with homologous proteins

图8 CaHSP21和其他植物同源蛋白系统进化分析

Fig.8 Phylogenetic analysis of CaHSP21 with its homologues from other species

表2 CaHSP21启动子顺式作用元件分析

Tab.2 Upstream elements in CaHSP21 promoter

元件名称 Name of element	序列(5'-3') Sequence(5'-3')	功能注释 Function annotation	数量 Amount
G-box	CACGTC	参与光反应	5
TC-rich repeats	GTTTTCTTAC	参与防御和应激反应	1
TGACG-motif	TGACG	茉莉酸甲酯响应元件	2
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件	2
LTR	CCGAAA	参与低温反应	1
ABRE	ACGTG	参与脱落酸反应	4

图9 CaHSP21在不同组织中(A)和高温下(B)表达量分析不同小写字母表示表达量差异显著(P<0.05)。

Fig.9 Expression levels of CaHSP21 in different tissues(A)and under high-temperature stress(B) Different lowercase letters indicated significant differences in relative expression at the P<0.05.

图10 CaHSP21的亚细胞定位 GFP.绿色荧光;CHI.叶绿体自发荧光;DIC.明场;Merged.叠加。

Fig.10 Sucellular localization of CaHSP21 GFP.Green fluorescence field;CHI.Chloroplast autofluorescence field;DIC.Generation indicates field;Merged.Superposition field.

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辣椒CaHSP21基因的克隆及表达分析

Cloning and Expression Analysis of CaHSP21 Gene in Capsicum annuum

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