Cloning and Expression Analysis of CaHSP21 Gene in Capsicum annuum

doi:10.7668/hbnxb.20196169

Abstract

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

CLC Number:

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.

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Figures/Tables 12

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

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

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

Fig.3 Conservative domain analysis of CaHSP21 protein

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

Fig.5 Secondary structure prediction of CaHSP21 protein

Fig.6 Tertiary structure prediction of CaHSP21 protein

Fig.7 Sequence alignment of CaHSP21 protein with homologous proteins

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

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

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.

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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