金线莲<i>C4H</i>基因克隆与表达及在黄酮合成途径中的作用

doi:10.7668/hbnxb.20194639

摘要/Abstract

摘要：

为探究肉桂酸-4-羟化酶(C4H)对金线莲类黄酮含量积累的影响,以金线莲为研究对象,利用硝酸铝显法测定其不同组织的总黄酮含量;基于同源克隆技术和RACE技术从金线莲分离克隆出C4H基因(ArC4H,GenBank登录号ON455229);利用实时荧光定量PCR (qRT-PCR)分析金线莲不同组织C4H基因表达量。结果表明,金线莲不同组织黄酮类物质含量有显著差异(叶>茎>根),其中叶片的总黄酮含量是根和茎的3倍多;系统进化树分析,发现金线莲与大多数兰科植物的亲缘关系比较接近,ArC4H基因的开放阅读框为1 518 bp,编码505个氨基酸,属于细胞色素P450超家族成员之一,理论分子量为65.17 ku,等电点为9.21,属于亲水蛋白;ArFLS具有序列保守性,在不同组织中均有表达,qRT-PCR分析表明,该基因在茎中表达量最高,根中次之,在叶中表达量极低,且总黄酮含量也有相似的趋势。据此初步确定,该基因为金线莲ArC4H基因。

关键词: 金线莲, C4H基因, 基因克隆, 表达分析, 黄酮

Abstract:

In order to investigate the effect of cinnamic acid 4-hydroxylase(C4H)on the accumulation of flavonoids in Anoectochilus roxburghii,the total flavonoid content of different tissues of A.roxburghii was determined by using the aluminium nitrate assay.Based on homologous cloning and RACE techniques,the C4H gene(ArC4H,GenBank accession number ON455229)was isolated and cloned from A.roxburghii;the expression of the C4H gene in different tissues of A.roxburghii was analysed by Real-time Quantitative PCR(qRT-PCR).The results showed that the flavonoid contents of different tissues of A.roxburghii differed significantly(leaf>stem>root),with the total flavonoid content of leaf being more than three times that of root and stem;the phylogenetic tree analysis revealed that A.roxburghii was close to most orchids and the open reading frame of the ArC4H gene was 1 518 bp,encoding 505 amino acids,and belonged to the cytochrome P450 superfamily,with a theoretical molecular weight of 65.17 ku and an isoelectric point of 9.21,which belonged to hydrophilic proteins;ArFLS had sequence conservatism,and was expressed in different tissues.qRT-PCR analysis showed that the gene had the highest expression in stems,followed by roots,and the expression in leaves was extremely low,and the total flavonoid content also showed a similar trend.Accordingly,the gene was initially identified as ArC4H gene.

Key words: Anoectochilus roxburghii, C4H gene, Gene cloning, Expression analysis, Flavonoid

林宏峻, 吕珂, 潘萍萍, 董莉莉, 徐志浩, 王忠华. 金线莲C4H基因克隆与表达及在黄酮合成途径中的作用[J]. 华北农学报, 2024, 39(S1): 31-38. doi: 10.7668/hbnxb.20194639.

LIN Hongjun, LÜ Ke, PAN Pingping, DONG Lili, XU Zhihao, WANG Zhonghua. Cloning and Expression of the Anoectochilus roxburghii C4H Gene and Its Role in the Flavonoid Synthesis Pathway[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 31-38. doi: 10.7668/hbnxb.20194639.

http://www.hbnxb.net/CN/Y2024/V39/IS1/31

图/表 13

图1 保守区序列

Fig.1 Conserved region sequence

图2 3'RACE和 5'RACE端序列 1.阴性对照;2.中间片段序列;3.5'端序列;4.3'端序列。

Fig.2 3'RACE and 5'RACE sequence 1.Negative control;2.Middle fragment sequence;3.5'end sequence;4.3'end sequence.

图3 氨基酸序列的同源性比对阴影部分:每段核酸序列中,该部分与其他序列一致。

Fig.3 Homology alignment of amino acid sequences Shaded portion:in each nucleic acid sequence,the portion is consistent with the rest of the sequence.

图4 ArC4H的系统进化树

Fig.4 Phylogenetic tree of ArC4H

图5 蛋白功能结构域分析

Fig.5 Analysis of protein functional structural domains

表1 金线莲ArC4H基因编码蛋白质的基本理化性质

Tab.1 Basic physicochemical properties of the proteins encoded by the ArC4H gene of Anoectochilus roxburghii

类别 Category	数量 Amount	类别 Category	数量 Amount
氨基酸数	572	相对分子量	65.167 23 ku
Number of amino acids		Molecular weight
理论等电点Theoretical pI	9.21	分子式Formula	C₂₉₈₈H₄₇₀₉N₇₉₃O₈₀₁S₁₉
脂肪指数Aliphatic index	102.40	原子总数Total number of atoms	9 310
亲水性的平均值Grand average of hydropathicity	-0.044	不稳定指数The instability index	44.07
带正电荷的残基总数(g+Lys)	74	带负电荷的残基总数(Asp+Glu)	64
Positively charged residues		Negatively charged residues

图6 蛋白疏水性分析

Fig.6 Protein hydrophobicity analysis

图7 蛋白质跨膜区 Y轴紫色线条表示不在结构细胞膜上;蓝色线条表示在结构细胞膜上;X轴红色线条为跨膜螺旋结构。

Fig.7 Protein transmembrane region Purple lines on the Y-axis indicate that they are not on the structural cell membrane;blue lines indicate that they are on the structural cell membrane;red lines on the X-axis are transmembrane helical structures.

图8 磷酸化位点红色.丝氨酸;绿色.苏氨酸;蓝色.酪氨酸;紫色.阈值评估。只有超过阈值才算是位点。

Fig.8 Phosphorylation sites Red.Serine;Green.Threonine;Blue.Tyrosine;Purple.Threshold assessment.Only those above the threshold are considered loci.

图9 蛋白信号肽分析

Fig.9 Protein signal peptide analysis

图10 蛋白质二级结构蓝色.α-螺旋;绿色.β-折叠;黄色.无规则卷曲;红色.延伸链。

Fig.10 Protein secondary structure Blue.Alpha helix;Green.Beta turn;Yellow.Random coil;Red.Extended strand.

图11 蛋白质三级结构

Fig.11 Protein tertiary structure

图12 C4H基因相对表达量和总黄酮含量不同字母表示差异显著,P<0.05。

Fig.12 Relative expression and total flavonoid content of C4H gene Different letters indicate significant differences,P<0.05.

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金线莲C4H基因克隆与表达及在黄酮合成途径中的作用

Cloning and Expression of the Anoectochilus roxburghii C4H Gene and Its Role in the Flavonoid Synthesis Pathway

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金线莲C4H基因克隆与表达及在黄酮合成途径中的作用

Cloning and Expression of the Anoectochilus roxburghii C4H Gene and Its Role in the Flavonoid Synthesis Pathway

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