Cloning， Expression Characteristics and Functional Verification of the Pepper CaFD1 Gene

doi:10.13560/j.cnki.biotech.bull.1985.2024-1249

Abstract

Abstract:

Objective The aim is to uncover the molecular mechanism of CaFD1 regulatying pepper’s flowering time and its function in the response to abiotic stress, thus providing gene resources for pepper molecular breeding. Method The AtFD homologous gene CaFD1 was obtained via RT-PCR from the cDNA of the stem of annual pepper ‘Zunla-1’. Its physical and chemical properties, protein structure, and phylogenetic connections were examined using bioinformatics. The gene’s expression traits under different tissues and stress treatments in pepper were analyzed by RT-qPCR. The gene silencing TRV-CaFD1 vector and subcellular localization 35S:CaFD1 vector were constructed and then introduced into pepper and tobacco respectively through Agrobacterium-mediated transformation. Result The entire genetic code of CaFD1, extending across 711 bases, was interpreted to form a protein containing 236 amino acids. The predicted molar mass was approximately 22.34 kD and demonstrated an isoelectric point, determined to be 7.71. It was a hydrophilic protein with an aliphatic amino acid index of 70.85 and high thermal stability. CaFD1 protein possessed two conserved domains, namely the basic amino acid region N-x9-R and the leucine zipper region x6-L-x6-L-x6-L. Its secondary structure was dominated by random coil with a minor portion of α‍-helix. Promoter prediction analysis revealed the existence of diverse cis-acting elements related to hormones and stress on the CaFD1 promoter. RT-qPCR confirmed that CaFD1 was mainly expressed in the stem, followed by the flower, and hardly expressed in the root. Subcellular localization indicated that the CaFD1 protein was localized in the nucleus. In virus-induced gene silencing plants, i.e., budding days and flowering days in the plants with silenced CaFD1 gene prolonged, while the phenotypes of overexpressing plants were complementary. Conclusion CaFD1 is predominantly expressed in the stem, has the function of promoting plant flowering, and responds to drought, high salt, ABA, and low light stress.

Key words: pepper, CaFD1, flowering time regulation, gene expression, functional verification, abiotic stress

PENG Shao-zhi, WANG Deng-ke, ZHANG Xiang, DAI Xiong-ze, XU Hao, ZOU Xue-xiao. Cloning， Expression Characteristics and Functional Verification of the Pepper CaFD1 Gene[J]. Biotechnology Bulletin, 2025, 41(5): 153-164.

Figures/Tables 12

Table 1 Primer sequences and names used in this study

引物名称 Names of primer	引物序列 Sequence of primer （5′-3′）	引物用途 Purpose of primer
QC-CaFD1_F	ATGTGGTCATCAAGCAGTGACAATAG	CaFD1基因克隆
QC-CaFD1_R	TCAAAATGGGGCGGTTGACG
GBD-CaFD1_F	ATACGTCCTAATCCCATGGAAGAAGTGTGG	35S：CaFD1载体
GBD-CaFD1_R	CCCTTGCTCACCATGGTACCAAATGGGGCGGTTGACGC
VIGS-CaFD1_F	TAAGGTTACCGAATTCATGGAAGAAGTGTGGAAAGAC	TRV-CaFD1载体
VIGS-CaFD1_R	GCTCGGTACCGGATCCTACTACTTGACAACTATCAGTT
Q-CaFD1_F	AGAACCGTGAGTCTGCTGCT	RT-qPCR
Q-CaFD1_R	TCTTGAGCCTGGCATTCTCT
UBI_F	CCACCTCTTCACTCTCTGCTCT	RT-qPCR
UBI_R	ACTAGGAAAAAACGCCCTTGGT
RT1_F	TGGCAGGATATATTGTGGTG	TRV_35S：CaFD1_OE载体
RT1_Overlap_R	CCACACTTCTTCCATGGGATTAGGACGTATC
RT2_Overlap_F	ATACGTCCTAATCCCATGGAAGAAGTGTGG
RT2_Overlap_R	CACGTCCTTAAATCCTTACTTGTACAGCTC
RT3_Overlap_F	GAGCTGTACAAGTAAGGATTTAAGGACGTGAACTCTG
RT3_R	GTTTACCCGCCAATATATCCTG

Fig. 1 Cloning (A), chromosome localization (B) and gene structure (C, D) analysis of gene CaFD1

Fig. 2 Analysis of the CaFD1 protein's physicochemical characteristicsA: Forecasting the hydrophilicity profile for the CaFD1 protein. B: Visual representation of the CaFD1 protein's predicted transmembrane regions. C: Predictive evaluation of the CaFD1 protein's signal peptide. D: Projection of the CaFD1 protein's potential phosphorylation locations

Fig. 3 Sequence alignment of CaFD1 and bZIP proteins from other plantsRed, purple and blue indicate 100%, >75%, and >50% similarity, respectively; Ca: Capsicum annuum L.; At: Arabidopsis thaliana; Os: Oryza sativa

Fig. 4 Phylogenetic analysis of bZIP proteins in different plants

Fig. 5 Prediction of secondary structures (A) and tertiary structures (B) of CaFD1 protein

Table 2 Statistics of cis-acting elements of CaFD1

顺式元件类型Cis-acting element	生物学功能Biological function
TGACG	茉莉酸甲酯Methyl jasmonate
I-box	光响应Light response
TCT	光响应Light response
CAT-box	分生组织表达Meristem expression
MBS	干旱诱导性Drought inducibility
TCA	水杨酸Salicylic acid
P-box	赤霉素Gibberellin
G-box	光响应Light response
Box 4	光响应Light response
G-Box	光响应Light response
TC	防御和应激反应Defense and stress response
MRE	光响应性Light responsiveness
TATC-box	赤霉素Gibberellin
ABRE	脱落酸Abscisic acid
CGTCA	茉莉酸Jasmonic acid
CAAT-box	启动子和增强子Promoter and enhancer
TATA-box	核心启动子元件Core promoter element

Fig. 6 Investigation into the tissue-specific expression profile of CaFD1

Fig. 7 Subcellular localization of CaFD1 gene

Fig. 8 CaFD1 is a flowering-promoting factorA: Flowering phenotype of CaFD1-silenced plants and CaFD1-overexpressed plants. B: Statistics on silencing efficiency, bud-emerging days and flowering days of CaFD1 plants and CaFD1-overexpressed plants

Fig. 9 Differential gene expression and regulatory network associated with CaFD1 gene silencing in pepper

Fig. 10 Relative expressions of CaFD1 under different stresses

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