辣椒CabZIP42的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0465

摘要/Abstract

摘要：

【目的】探究辣椒bZIP基因在响应非生物胁迫应答过程中的作用，为提高辣椒抗逆性提供基因资源。【方法】以辣椒‘G7’为材料，基于前期转录组测序得到一个候选基因CabZIP42，并对其进行克隆，通过生物信息学的方法对其编码蛋白的分子特征进行分析，并利用RT-qPCR技术分析其在不同组织、不同胁迫处理下的表达模式。【结果】CabZIP42编码区全长1 233 bp，编码410个氨基酸，预测分子量为44.85 kD，理论等电点为9.47；属于亲水蛋白，脂肪族氨基酸指数为61.88，热稳定性较高。CabZIP42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。该蛋白二级结构以无规则卷曲为主，还含有少量的α-螺旋。系统进化分析显示，CabZIP42与番茄、马铃薯的蛋白进化关系最近。启动子预测分析表明，CabZIP42启动子上存在多种激素和胁迫相关的顺式作用元件。【结论】CabZIP42在辣椒叶片中的表达量最高，并且参与ABA信号转导，响应干旱、高温与高盐胁迫。

关键词: 辣椒, bZIP转录因子, 克隆, 表达分析, 非生物胁迫

Abstract:

【Objective】To explore the role of bZIP gene in response to abiotic stress would provide genetic resources for improving the stress resistance of peppers. 【Method】A candidate gene CabZIP42 was obtained and cloned based on transcriptome sequencing in pepper ‘G7’, the molecular characteristics of the encoded protein were analyzed by bioinformatics methods, and its expression patterns were analyzed by RT-qPCR in different tissues and under different stress treatments.【Result】The coding region of CabZIP42 was 1 233 bp in length, encoding 410 amino acids, with a predicted molecular weight of 44.85 kD and a theoretical isoelectric point of 9.47. The CabZIP42 protein contained two conserved domains, the basic amino acid region N-x9-R and the leucine zipper region x6-L-x6-L-x6-L. The secondary structure of the protein was dominated by random coils, and contained a small amount of α-helix. Phylogenetic analysis showed that CabZIP42 had the most recent protein evolutionary relationship with tomato and potato. Promoter prediction analysis showed that there were multiple hormonal and stress-related cis-acting elements on the CabZIP42 promoter.【Conclusion】The expression of CabZIP42 gene was the highest in pepper leaves, and it was involved in ABA signal transduction in response to drought, high temperature and high salt stress.

Key words: pepper, bZIP transcription factor, cloning, expression analysis, abiotic stress

李白雪, 李金玲, 陈春林, 杜清洁, 李猛, 王吉庆, 马勇斌, 肖怀娟. 辣椒CabZIP42的克隆及表达分析[J]. 生物技术通报, 2024, 40(12): 93-101.

LI Bai-xue, LI Jin-ling, CHEN Chun-lin, DU Qing-jie, LI Meng, WANG Ji-qing, MA Yong-bin, XIAO Huai-juan. Cloning and Expression Analysis of CabZIP42 Gene in Pepper[J]. Biotechnology Bulletin, 2024, 40(12): 93-101.

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参考文献 33

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基因 Gene	引物序列 Primer sequence（5'-3'）	用途 Purpose
CabZIP42	F: ATGGGATCTTACATGAACTTC R: CTACCAAGGCCCTGTCAGT	克隆 Cloning
	F: CTCGGCCACTTAGGCAGAAA R: CTAACTACCCCGGCCTTCAC	实时荧光定量 RT-qPCR
CaUBI	F: TGTCCATCTGCTCTCTGTTG R: CACCCCAAGCACAATAAGAC	内参基因 Reference gene

基因 Gene	引物序列 Primer sequence（5'-3'）	用途 Purpose
CabZIP42	F: ATGGGATCTTACATGAACTTC R: CTACCAAGGCCCTGTCAGT	克隆 Cloning
	F: CTCGGCCACTTAGGCAGAAA R: CTAACTACCCCGGCCTTCAC	实时荧光定量 RT-qPCR
CaUBI	F: TGTCCATCTGCTCTCTGTTG R: CACCCCAAGCACAATAAGAC	内参基因 Reference gene

顺式元件类型Cis-acting element	生物学功能Biological function
STRE	防御应激与胁迫响应元件Element involved in defensive stress and stress response
CAAT-box	启动子和增强子Promoter and enhancer
AE-box	光响应Light responsiveness
BOX 4	光响应Light responsiveness
G-box	光响应Light responsiveness
CGTCA-motif	茉莉酸甲酯响应元件Element involved in MeJA responsiveness
TGACG-motif	茉莉酸响应元件Element involved in jasmonic acid responsiveness
ERE	乙烯响应元件Element involved in ethylene responsiveness
TCA	水杨酸响应元件Element involved in SA responsiveness
TCA-element	水杨酸响应元件Element involved in SA responsiveness
as-1	水杨酸响应的顺式作用元件cis-acting element involved in SA responsiveness
MYB	MYB识别和结合元件MYB identifying and binding elements
MYC	MYC识别和结合元件MYC identifying and binding elements
Myb	Myb识别和结合元件MYb identifying and binding elements
CAT-box	分生组织表达相关元件Elements related to meristem expression
F-box	防御应激与胁迫响应元件Element involved in defense stress and stress response
LAMP-element	环境响应元件Element involved in environment responsiveness
TCT-motif	光周期响应元件Element involved in photoperiod responsiveness
TGA-element	植物生长激素响应元件Element involved in plant growth hormone responsiveness
circadian	昼夜节律响应元件Element involved in circadian responsiveness
AAGAA-motif	脱落酸响应元件ABA-responsive element
ABRE	脱落酸响应元件ABA-responsive elements
GATA-motif	光响应Light responsiveness
GT1-motif	光响应Light responsiveness
TC-rich repeats	防御应激与胁迫响应元件Element involved in defensive stress and stress response
TATA-box	转录起始-30左右的核心启动子元件Core promoter element around -30 of transcription start

顺式元件类型Cis-acting element	生物学功能Biological function
STRE	防御应激与胁迫响应元件Element involved in defensive stress and stress response
CAAT-box	启动子和增强子Promoter and enhancer
AE-box	光响应Light responsiveness
BOX 4	光响应Light responsiveness
G-box	光响应Light responsiveness
CGTCA-motif	茉莉酸甲酯响应元件Element involved in MeJA responsiveness
TGACG-motif	茉莉酸响应元件Element involved in jasmonic acid responsiveness
ERE	乙烯响应元件Element involved in ethylene responsiveness
TCA	水杨酸响应元件Element involved in SA responsiveness
TCA-element	水杨酸响应元件Element involved in SA responsiveness
as-1	水杨酸响应的顺式作用元件cis-acting element involved in SA responsiveness
MYB	MYB识别和结合元件MYB identifying and binding elements
MYC	MYC识别和结合元件MYC identifying and binding elements
Myb	Myb识别和结合元件MYb identifying and binding elements
CAT-box	分生组织表达相关元件Elements related to meristem expression
F-box	防御应激与胁迫响应元件Element involved in defense stress and stress response
LAMP-element	环境响应元件Element involved in environment responsiveness
TCT-motif	光周期响应元件Element involved in photoperiod responsiveness
TGA-element	植物生长激素响应元件Element involved in plant growth hormone responsiveness
circadian	昼夜节律响应元件Element involved in circadian responsiveness
AAGAA-motif	脱落酸响应元件ABA-responsive element
ABRE	脱落酸响应元件ABA-responsive elements
GATA-motif	光响应Light responsiveness
GT1-motif	光响应Light responsiveness
TC-rich repeats	防御应激与胁迫响应元件Element involved in defensive stress and stress response
TATA-box	转录起始-30左右的核心启动子元件Core promoter element around -30 of transcription start