杂交兰ChChlH基因的克隆及其表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1359

摘要/Abstract

摘要：

目的克隆获得杂交兰镁螯合酶H亚基基因（ChlH），并对其表达水平和功能进行分析，为探究ChlH基因在杂交兰叶色变异中的调控作用提供重要依据。方法利用RT-PCR和RACE技术从杂交兰叶片中克隆获得ChChlH基因序列，对其进行生物信息学分析；利用基因组步移法获得ChChlH基因启动子序列，分析其顺式作用元件；利用RT-qPCR法分析ChChlH在不同组织及不同品种叶片中的表达情况；基于VIGS技术验证沉默ChChlH基因对烟草叶色变化的影响。结果 ChChlH基因全长4 321 bp，编码区长4 155 bp，GenBank登录号为OL741687.1，编码1 384个氨基酸；ChChlH蛋白可能是一种亲水性不稳定蛋白质，无信号肽和跨膜结构，属于PLN03069超级家族；ChChlH与铁皮石斛ChlH亲缘关系较近，其中铁皮石斛ChlH与ChlI和ChlD发生蛋白互作；ChChlH启动子包含光响应及脱落酸、乙烯、赤霉素、水杨酸、茉莉酸甲酯信号响应元件，具有MYB、WRKY结合位点；ChChlH在叶中表达量较高，且绿叶区域中的表达量显著高于叶艺区域叶片中的表达量。沉默ChChlH后烟草老叶中的光合色素含量及ChlH基因的相对表达量显著降低。结论明确了ChChlH基因的序列和表达特征模式，该基因可能在叶绿素合成和叶色变异中发挥重要作用。

关键词: 杂交兰, 镁螯合酶H亚基基因, 基因克隆, 生物信息学分析, 表达分析

Abstract:

Objective The magnesium chelatase H subunit gene (ChlH) was cloned from Cymbidium hybrid, and its expression patterns and function were analyzed, thereby providing important insights into the regulatory role of the ChlH gene in leaf color variation in Cymbidium hybrid. Method The ChChlH gene sequence was cloned by RT-PCR and RACE, followed by bioinformatics analysis. The promoter sequence of the ChChlH gene was obtained via genome walking, and its cis-acting elements were analyzed. The expression patterns of ChChlH in different tissues and different cultivars were examined using RT-qPCR. Furthermore, the effect of ChChlH silencing on leaf color variation in Nicotiana tabacum was verified using a virus-induced gene silencing (VIGS) system. Result The full-length sequence of ChChlH is 4 321 bp, with a coding region of 4 155 bp encoding 1 384 amino acids, and its GenBank accession number is OL741687.1. Bioinformatics analysis indicates that the ChChlH protein might be a hydrophilic and unstable protein, lacking a signal peptide and transmembrane domains, and belongs to the PLN03069 superfamily. ChChlH showed high affinity with the ChlH protein from Dendrobium catenatum, which is known to interact with ChlI and ChlD. Promoter analysis revealed that the ChChlH promoter contains light-responsive elements, as well as cis-acting elements involved in responses to abscisic acid, ethylene, gibberellin, salicylic acid, and methyl jasmonate, along with MYB and WRKY transcription factor binding sites. Expression analysis showed that ChChlH was highly expressed in leaves, with significantly higher transcript levels in green leaf areas than in variegated regions. Silencing of ChChlH in N. tabacum led to a significant reduction in photosynthetic pigment content and ChlH expression in older leaves. Conclusion The study clarified the sequence and expression pattern of the ChChlH gene, which may play an important role in chlorophyll synthesis and the variation of leaf color.

Key words: Cymbidium hybrid, magnesium chelatase H subunit gene, gene cloning, bioinformatics analysis, expression analysis

林榕燕, 孔兰, 吴建设, 林兵, 钟淮钦. 杂交兰ChChlH基因的克隆及其表达分析[J]. 生物技术通报, 2026, 42(10): 229-237.

LIN Rong-yan, KONG Lan, WU Jian-she, LIN Bing, ZHONG Huai-qin. Cloning and Expression Analysis of ChChlH Gene in Cymbidium hybrid[J]. Biotechnology Bulletin, 2026, 42(10): 229-237.

图/表 6

表1 杂交兰ChlH基因克隆及RT-qPCR所用引物

Table 1 Primers used in ChlH clone and RT-qPCR in Cymbidium hybrid

引物名称 Primer Name	引物序列 Primer sequence	作用 Function
AP	5′-GGCCACGCGTCGACTAGTAC（T）₁₇-3′	逆转录引物 Reverse transcription primer
AAP	5′-GGCCACGCGTCGACTAGTAC（G）₈-3′	锚定引物 Anchored primers
AUAP	5′-GGCCACGCGTCGACTAGTAC-3′	锚定引物 Anchored primers
ChlH-F	5′-CAATGGCTTCTCTGGTTTTCACTC-3′	保守区扩增引物 Conserved fragment amplification primers
ChlH-R	5′-TTAGCGCTCAATACCCTCGATCT-3′	保守区扩增引物 Conserved fragment amplification primers
ChlH-3′-1	5′-GAATCTCGTACAGTCACTTAGGCGCGA-3′	3′ RACE 引物 3′ RACE primers
ChlH-3′-2	5′-TATGAGGGGATGATGAGGAGC-3′	3′ RACE 引物 3′ RACE primers
ChlH-5′-1	5′-AGAAGGTGCGGTAAGTGGTCTCGTC-3′	5′ RACE 引物 5′ RACE primers
ChlH-5′-2	5′-CGTTGCAGAAGAAGACCCTTGAT-3′	5′ RACE 引物 5′ RACE primers
ChlH-ORF-F	5′-ACTGCCCGCGATCACTTCCTCAGC-3′	ORF扩增引物 ORF amplification primers
ChlH-ORF-R	5′-CAAAAATACAAACAGTCCAACTCCTG-3′	ORF扩增引物 ORF amplification primers
ChlH-SP1	5′-CGAAGATGAGCGAGCCGATGAATACG-3′	启动子序列引物 Promoter sequence primers
ChlH-SP2	5′-GACTGCGGCGGATAGGGAGGATTGGTACTG-3′
ChlH-SP3	5′-TTGATGTGGGTTGTTGGGTGAGGCGGGGAAGG-3′
ChACT -F	5′-GGGTGCTTATGTTGGTGATG-3′	内参基因引物 For the internal control
ChACT -R	5′-TTCAGAGGGGCTTCAGTAAGG-3′	内参基因引物 For the internal control
ChlH-RT-F	5′-TTCAGCAGCCCTAACCTCAACGTCGCT-3′	RT-qPCR引物（杂交兰） RT-qPCR（Cymbidium hybrid）
ChlH-RT-R	5′-GAAGGAGTAGTAGGCGGCAAAGCCATGGTG-3′	RT-qPCR引物（杂交兰） RT-qPCR（Cymbidium hybrid）
ChChlH-VIGS-F	5′-CGACGACAAGACCGTCACCCCCGGTGAACAACAAGCCTTTCGT-3′	VIGS引物 VIGS primers
ChChlH-VIGS-R	5′-GAGGAGAAGAGCCGTCGGCCCATCTTATAGCTCTTGTACATAG-3′	VIGS引物 VIGS primers
ChChlH-RT-F	5′-CCCGGTGAACAACAAGCCTTTCGT-3′	RT-qPCR引物（烟草） RT-qPCR（N. tabacum）
ChChlH-RT-R	5′-GCATCCCTGCCGGAAAACACTATCGG-3′	RT-qPCR引物（烟草） RT-qPCR（N. tabacum）

图1 杂交兰ChlH序列扩增产物及启动子作用元件预测A：ChlH序列扩增产物（M1：DL 2 000 Marker；M2：DL 5 000 Marker；1：保守区扩增条带；2：3′ RACE扩增条带；3：5′ RACE扩增条带；4-6：ORF验证条带）；B：ChChlH启动子序列扩增产物（7：启动子序列扩增条带）；C：ChChlH启动子作用元件预测

Fig. 1 Amplification products of ChlH gene from Cymbidium hybrid and prediction of its promoter cis-acting elementsA: Amplification product of ChlH sequence (M1: DL 2 000 Marker; M2: DL 5 000 Marker; 1: conserved region amplification band; 2: 3′ RACE amplification band; 3: 5′ RACE amplification band; 4-6: ORF verification bands). B: Amplification product of the ChChlH promoter sequence (7: promoter amplification band). C: Promoter acting elements prediction of ChChlH

图2 杂交兰ChChlH蛋白亲/疏水性（A）、磷酸化位点（B）、二级结构（C）和保守结构域（D）预测

Fig. 2 Prediction of the hydrophilicity/hydrophobicity (A), phosphorylation sites (B), secondary structure (C), and conserved domain structure (D) of ChChlH in Cymbidium hybrid

图3 杂交兰ChlH系统进化树分析（A）和蛋白质互作分析（B）

Fig. 3 Phylogenetic tree analysis (A) and protein interaction analysis (B) of Cymbidium hybird ChlH

图4 杂交兰ChChlH基因的表达情况分析不同字母表示显著性差异（P<0.05），下同

Fig. 4 Expression analysis of ChChlH in Cymbidium hybridDifferent letters indicate significant differences (P<0.05), the same below

图5 重组载体的构建及ChChlH基因沉默植株的效果分析A：重组载体 pTRV2-ChChlH扩增产物（M：DL 2 000 Marker）；B：注射pTRV2-ChChlH载体后的烟草表型；C：注射不同载体后烟草叶片光合色素含量；D：注射不同载体后ChChlH基因的相对表达量

Fig. 5 Construction of recombinant vectors and the effect analysis of ChChlH gene silencing plantsA: Amplification product of the recombinant vector pTRV2-ChChlH (M: DL 2 000 Marker). B: Phenotype of tobacco after the pTRV2-ChChlH vector injection. C: Content of photosynthetic pigments in tobacco leaves after different vector injection. D: Relative expression of ChChlH gene after different vectors injection

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