Cloning and Expression Analysis of RsCHS1 Gene in Red Radish（Raphanus sativus L.)

doi:10.15933/j.cnki.1004?3268.2023.09.012

Abstract

Abstract: In order to explore the role of RsCHS1 gene in pigment accumulation of Raphanus sativus L.，the open reading frame（ORF） of RsCHS1 gene was cloned from fleshy root of red radish.The physicochemical properties of RsCHS1 protein were analyzed by bioinformatics.In addition，the expressions of RsCHS1 gene in the leaves，fleshy roots and fleshy root skins of Raphanus sativus L.at vegetative stage，and in the flowers，pod and fleshy roots at flowering stage were analyzed using real‑time quantitative PCR（qRT‑PCR）.What’s more，the function of RsCHS1 gene was verified through the overexpression in Arabidopsis thaliana.The results showed that two RsCHS1 genes were cloned.The ORF sequence of RsCHS1‑a gene was 765 bp，which encoded 254 amino acids，while the ORF sequence of RsCHS1‑b gene was 966 bp，which encoded 321 amino acids.Secondary and tertiary structure analysis showed that α‑helix was the main structural element of RsCHS1 protein，while β‑angle，lamellar structure and random curling were distributed throughout the protein.qRT‑PCR analysis illuminated that the expression level of RsCHS1‑a gene in fleshy root（26.8），root bark（32.4）and flower（6.7）were higher than that in leaf（1.0）and fruit（0.4）.The expression level of RsCHS1‑b gene in fleshy root（41.4），root bark（41.9）and flower（9.7）were higher than that in leaf（1.0）and fruit（0.6），indicating that RsCHS1 gene exhibited a tissue specific expression.Through the transformation of RsCHS1 gene into Arabidopsis plants，it was found that the petiole and stem of transgenic plant accumulated a large amount of anthocyanins，indicating that RsCHS1 gene plays a predominant role in the anthocyanin accumulation in red radish.

Key words: Raphanus sativus L., Chalcone synthase gene（CHS）, Cloning, Expression pattern, Anthocyanin

摘要： 为解析RsCHS1基因在胭脂萝卜色素积累中的作用，以胭脂萝卜肉质根肉质cDNA为模板，同源克隆了RsCHS1基因的开放阅读框（ORF），采用生物信息学方法对RsCHS1蛋白的理化性质进行分析。利用实时荧光定量PCR（qRT-PCR）分析了RsCHS1基因在胭脂萝卜营养期叶、肉质根肉质和肉质根皮及花期花、荚果和肉质根肉质中的表达情况，同时构建RsCHS1基因过表达载体，异源转化拟南芥，进行RsCHS1基因功能验证。结果表明，克隆得到2个RsCHS1基因，其中RsCHS1-a基因ORF序列长765 bp，编码254个氨基酸；RsCHS1-b基因ORF序列长966 bp，编码321个氨基酸。编码蛋白二、三级结构预测显示，α-螺旋是RsCHS1主要结构元件，β-转角、片层结构、无规则卷曲分布于整个蛋白质中。qRTPCR结果表明，RsCHS1-a基因在肉质根肉质（26.8）、根皮（32.4）和花（6.7）中的相对表达量比叶（1.0）和果实（0.4）中高，RsCHS1-b基因在肉质根肉质（41.4）、根皮（41.9）和花（9.7）中的相对表达量比叶（1.0）和果实（0.6）中高，说明RsCHS1基因具有组织表达特异性。在拟南芥中过表达RsCHS1基因，发现转基因植株叶柄和茎累积大量花青素，表明RsCHS1基因在胭脂萝卜花青素积累中起到重要作用。

关键词: 胭脂萝卜, 查尔酮合成酶基因（CHS）, 克隆, 表达模式, 花青素

CLC Number:

S631.1

王川艺, 黄红涛, 陈发波, 郑张飞, 方平. 胭脂萝卜RsCHS1 基因克隆及表达分析[J]. 河南农业科学, 2023, 52(9): 122-132.

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