辣椒CaFD1基因克隆、表达特征及功能验证

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

摘要/Abstract

摘要：

目的探究CaFD1调控辣椒开花时间的分子机制，以及其在应对非生物胁迫反应中的作用，为辣椒的分子育种提供可用的基因资源。方法利用RT-PCR技术，从一年生辣椒（Capsicum annuum L.）‘遵辣1号’茎器官的cDNA中克隆出AtFD同源基因CaFD1。借助生物信息学手段，对该基因的理化性质、蛋白结构以及系统进化关系展开分析。利用RT-qPCR技术研究基因在辣椒不同组织以及不同胁迫处理下的表达特征。构建基因沉默载体TRV-CaFD1、过表达载体TRV-CaFD1-OE和亚细胞定位35S∶CaFD1载体，再通过农杆菌侵染法分别转化辣椒和烟草。结果 CaFD1的ORF长度为711 bp，所含氨基酸数量达236个，预估的分子量约为22.34 kD，理论上的等电点值为7.71。该蛋白属于亲水性蛋白，脂肪族氨基酸指数为70.85，热稳定性较高。CaFD1蛋白中包含2个保守结构域，碱性氨基酸区域N-x9-R和亮氨酸拉链区x6-L-x6-L-x6-L。其二级结构以无规则卷曲为主要特征，还包含少量的α-螺旋。启动子预测分析表明，CaFD1启动子上存在多种激素和胁迫相关的顺式作用元件。RT-qPCR证实CaFD1主要在茎中表达，其次是花，在根中几乎不表达。亚细胞定位实验说明CaFD1蛋白定位在细胞核中。在病毒诱导的基因沉默植株中，沉默CaFD1基因植株出现现蕾天数和开花天数延长的表型，而过表达植株的表型则得到互补。结论 CaFD1在茎中优势表达，能够促进植物开花，并且对干旱、高盐、ABA与弱光胁迫做出响应。

关键词: 辣椒, CaFD1, 开花时间调控, 基因表达, 功能验证, 非生物胁迫

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

彭绍智, 王登科, 张祥, 戴雄泽, 徐昊, 邹学校. 辣椒CaFD1基因克隆、表达特征及功能验证[J]. 生物技术通报, 2025, 41(5): 153-164.

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.

图/表 12

表1 本研究所用引物序列和名称

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

图1 CaFD1基因克隆（A）、染色体定位（B）与结构分析（C、D）

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

图2 CaFD1蛋白理化性质分析A：CaFD1蛋白亲水性的预测结果；B：CaFD1蛋白跨膜结构域的预测展示；C：CaFD1蛋白信号肽的预测分析；D：CaFD1蛋白磷酸化位点的预测情形

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

图3 CaFD1与其他植物bZIP蛋白序列比对红色、紫色、蓝色分别代表100%、大于75%和大于50%的相似性；Ca：辣椒；At：拟南芥；Os：水稻

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

图4 不同植物bZIP蛋白的系统进化分析

Fig. 4 Phylogenetic analysis of bZIP proteins in different plants

图5 CaFD1蛋白二级结构（A）、蛋白三级结构（B）的预测

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

表2 CaFD1顺式作用元件统计

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

图6 CaFD1在不同组织中的特异性表达分析*： P<0.05； **： P<0.01； ***： P<0.001， the same below

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

图7 CaFD1基因亚细胞定位

Fig. 7 Subcellular localization of CaFD1 gene

图8 CaFD1是开花促进因子A：沉默CaFD1植株及过表达CaFD1植株开花表型；B：沉默CaFD1植株及过表达CaFD1植株的沉默效率、现蕾天数和开花天数统计

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

图9 辣椒CaFD1基因沉默相关基因表达差异与调控网络

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

图10 CaFD1在不同胁迫处理下的相对表达量

Fig. 10 Relative expressions of CaFD1 under different stresses

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