Cloning and Expression Analysis of ChChlH Gene in Cymbidium hybrid

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

Abstract

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

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

Figures/Tables 6

References 31

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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）

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）