观赏辣椒<i>ARP</i>基因克隆及表达分析

doi:10.7668/hbnxb.20194037

摘要/Abstract

摘要：

为研究ARP在观赏辣椒低温胁迫下的响应情况,旨在为后续观赏辣椒低温下基因功能研究提供理论基础。通过RT-PCR技术从观赏辣椒中克隆得到与辣椒、番茄、马铃薯的同源ARP基因,命名为CfARP,分析其在观赏辣椒不同组织以及低温处理下的表达情况;通过利用生物信息学分析进行基因蛋白编码情况、理化性质、基因亲缘关系研究,并利用qRT-PCR技术检测CfARP在观赏辣椒不同组织以及低温处理下的表达量。结果表明,CfARP基因CDS序列为342 bp,与辣椒(遵辣1号)同源性为100%,可编码113个氨基酸,含有一个保守的Auxin-repressed结构域;蛋白理化分析发现,CfARP蛋白分子量为12.156 ku,等电点为10.22,亲水性平均系数为-0.913,初步预测CfARP为亲水性蛋白;与其他物种ARP氨基酸序列对比发现,CfARP在茄科植物中高度保守。实时荧光定量PCR结果显示,CfARP基因在观赏辣椒茎中表达量最高,根中次之,叶和花中相对较少;CfARP基因的表达量随着低温处理时长的增加而不断升高。初步推测CfARP在观赏辣椒响应低温胁迫过程中具有一定作用。

关键词: 观赏辣椒, CfARP, 基因克隆, 生物信息学分析, qRT-PCR, 低温胁迫

Abstract:

In order to explore the response of ARP to low temperature stress in ornamental pepper,and provide a theoretical basis for subsequent research on gene function in ornamental pepper under low temperature stress.This study cloned homologous ARP gene from ornamental pepper using RT-PCR technology,named CfARP,and analyzed their expression in different tissues of ornamental peppers and under low temperature treatment;Using bioinformatics analysis to study gene protein coding,physicochemical properties,and genetic relationships,and using qRT-PCR technology to detect the expression level of CfARP in different tissues of ornamental pepper and under low temperature treatment.The results showed that the CDS sequence of the CfARP gene was 342 bp,with 100% homology with Zunla 1.It could encode 113 amino acids and contained a conserved Auxin-repressed domain;protein physicochemical analysis revealed that the molecular weight of CfARP protein was 12.156 ku,its isoelectric point was 10.22,and the average coefficient of hydrophilicity was -0.913.It was preliminarily predicted that CfARP was a hydrophilic protein;compared with other species' ARP amino acid sequences,it was found that CfARP was highly conserved in Solanaceae plants.The Real-time Fluorescence Quantitative PCR results showed that the expression level of CfARP gene was the highest in the stems of ornamental pepper,followed by roots,and relatively less in leaves and flowers;the expression level of CfARP gene continuously increased with the increase of low-temperature treatment duration.It was preliminarily speculated that CfARP had a certain role in the response of ornamental pepper to low temperature stress.

Key words: Capsicum annuum L.var.fasciculatum, CfARP, Gene cloning, Bioinformatics analysis, qRT-PCR, Cold stress

黄渺, 罗悠悠, 倪芮, 肇瑾. 观赏辣椒ARP基因克隆及表达分析[J]. 华北农学报, 2023, 38(5): 69-76. doi: 10.7668/hbnxb.20194037.

HUANG Miao, LUO Youyou, NI Rui, ZHAO Jin. Cloning and Expression Analysis of ARP Gene in Capsicum annuum L.var.fasciculatum[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 69-76. doi: 10.7668/hbnxb.20194037.

http://www.hbnxb.net/CN/Y2023/V38/I5/69

图/表 10

图1 番茄、马铃薯、辣椒ARP基因序列比对红框为引物序列。

Fig.1 Sequences alignment of CaARP,SlARP and StARP Primer sequences are marked in red.

图2 CfARP琼脂糖凝胶电泳

Fig.2 Agarose gel electrophoresis of the PCR products of CfARP M.DNA Marker;1,2.CfARP。

图3 CfARP基因及氨基酸序列

Fig.3 Gene and amino acid sequence of CfARP

图4 CfARP三级结构组成

Fig.4 Protein tertiary structure of CfARP

图5 ARP基因序列多重对比

Fig.5 Sequences alignment of ARP

图6 ARP氨基酸序列多重对比

Fig.6 Multiple alignment results of the amino acid sequence of ARP

图7 ARP基因序列系统发育树

Fig.7 Phylogenetic tree of ARP

图8 观赏辣椒CfARP结构域预测

Fig.8 The prediction of CfARP conserved domain

图9 CfARP在不同组织中的表达分析不同小写字母代表相对表达量在0.05水平上差异显著。图10同。

Fig.9 Expression analysis of CfARP in different organs Different lowercase letters represent significant differences in relative expression at the 0.05 level.The same as Fig.10.

图10 低温处理不同时长CfARP的表达分析

Fig.10 Expression analysis of CfARP at different time under low temperature treatment

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