Cloning and Expression Analysis of Mg-Chelatase H Subunit Gene CsChlH in Tea Plant（Camellia sinensis）

doi:10.16420/j.issn.0513-353x.2023-0128

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (1): 91-102.doi: 10.16420/j.issn.0513-353x.2023-0128

Cloning and Expression Analysis of Mg-Chelatase H Subunit Gene CsChlH in Tea Plant（Camellia sinensis）

YE Yufan¹^,², WANG Yujie¹, FU Qianyuan¹^,², WANG Lu¹, HAO Xinyuan¹, DING Changqing¹, WANG Xinchao¹, CAO Hongli²^,³^,^**(), LI Nana¹^,^**

¹ Key Laboratory of Biology，Genetics and Breeding of Special Economic Animals and Plants，Ministry of Agriculture and Rural Affairs，National Center for Tea Improvement，Tea Research Institute，Chinese Academy of Agricultural Sciences，Hangzhou 310008，China
² Key Laboratory of Tea Science at Universities in Fujian，College of Horticulture，Fujian Agriculture and Forestry University，Fuzhou 350002，China
³ College of Food Science，Southwest University，Chongqing 400715，China

Received:2023-04-25 Revised:2023-08-28 Online:2024-01-25 Published:2024-01-16
Contact: **（E-mail：nanali@tricaas.com，lili9885@126.com）

Abstract

Abstract:

In this study，the complete open reading frame sequence of CsChlH gene encoded 1 382 amino acid residues was cloned in tea plant（NCBI accession number：ON256194）. Phylogenetic tree analysis showed that CsChlH was closely related to AcChlH of kiwifruit. Promoter cloning and sequence analysis showed that CsChlH gene may respond to multiple signals such as light，drought，heat shock，low temperature，ABA，ethylene，and may regulate the metabolism of chlorophylls，flavonoids，sugars，as well as the development of chloroplast，mesophyll，guard cell and other organs. CsChlH protein was localized in the chloroplast. Tissue-specific expression analysis found that CsChlH gene was highly expressed in the chlorophyll-rich source tissues but lowly expressed in the chlorophyll-deficient sink tissues. The expression of CsChlH gene was down-regulated in response to long-term low light conditions，cold and drought stresses，while up-regulated by short-term ABA，ethylene and long-term heat stress. The results revealed that CsChlH gene played a vital role in the regulation of chlorophyll metabolism and stress resistance of tea plant.

Key words: Camellia sinensis, CsChlH, promoter, gene expression, abscisic acid, GUS enzyme activity

YE Yufan, WANG Yujie, FU Qianyuan, WANG Lu, HAO Xinyuan, DING Changqing, WANG Xinchao, CAO Hongli, LI Nana. Cloning and Expression Analysis of Mg-Chelatase H Subunit Gene CsChlH in Tea Plant（Camellia sinensis）[J]. Acta Horticulturae Sinica, 2024, 51(1): 91-102.

Figures/Tables 7

Table 1 Primers for cloning and expression analysis of CsChlH in tea plant

序列名称 Sequence name	上游引物序列（5′-3′） Forward primer	下游引物序列（5′-3′） Reverse primer	用途 Application
CsChlH ORF	CAGGAAACAGGTGAGTTTTCTGGAAG	TCCATCAAAGAGCAATAGAGAGATCAGG	扩增Amplification
CsChlH promoter	CACTACGAGGTTTACGAGATAAACAG	CTAAAGAAGCCATTGTCAATGCTCT	扩增Amplification
CsPTB ORF	ACCAAGCACACTCCACACTATCG	TGCCCCCTTATCATCATCCACAA	qRT-PCR
CsChlH ORF	TATGAGGGTGTGCGCGAAAT	GTCTCCCAATACCCACGTCC	qRT-PCR
CsChlH ORF	ACGAGAGTGTCGGAGCTCGGTACCATGGCTTCTTTAGTTTCTTCACCATTC	CTCGCCCTTGCTCACCATGTCGACACGATCAATCCCTTCAATCTTGTCTTC	亚细胞定位 Subcellular localization
CsChlH promoter	CAGGTCGACTCTAGAGGATCCCACTACGAGGTTTACGAGATAAACAG	CCTCAGATCTACCATGGTACCTGTCAATGCTCTCTCTGTTCTTCC	启动子连接GUS Promoter linked to GUS

Fig. 1 Open reading frame amplification（A），coding protein sequence（B）and gene structure（C）of CsChlH in tea plant The red underlined area is the protein conservative domain.

Fig. 2 PCR amplification（A）and sequence（B）of CsChlH promoter in tea plant

Fig. 3 Subcellular localization of CsChlH protein in tea plant A and E：GFP green fluorescence signal；B：RFP red fluorescence signal of nucleus；F：Chlorophyll autofluorescence；C and G：Bright field；D and H：Merged signal.

Fig. 4 Tissue-specific expression patterns of CsChlH in tea plant Different lowercase indicate significant differences（P < 0.05）. The same below.

Fig. 5 Expression levels of CsChlH under shading，phytomone，temperature，and drought treatments in tea plant * indicates significant differences between different treatments（P < 0.05）.

Fig. 6 GUS staining and enzyme activity detection of prCsChlH::GUS transgenic tobacco after drought treatment

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Cloning and Expression Analysis of Mg-Chelatase H Subunit Gene CsChlH in Tea Plant（Camellia sinensis）

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