茶树镁离子螯合酶H亚基基因CsChlH的克隆及其表达分析

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

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

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

茶树镁离子螯合酶H亚基基因CsChlH的克隆及其表达分析

叶玙璠¹^,²^,^*, 王誉洁¹^,^*, 傅前媛¹^,², 王璐¹, 郝心愿¹, 丁长庆¹, 王新超¹, 曹红利²^,³^,^**(), 李娜娜¹^,^**

¹ 中国农业科学院茶叶研究所，国家茶树改良中心，农业农村部特种经济动植物生物学与遗传育种重点实验室，杭州 310008
² 福建农林大学园艺学院，茶学福建省高校重点实验室，福州 350002
³ 西南大学食品科学学院，重庆 400715

收稿日期:2023-04-25 修回日期:2023-08-28 出版日期:2024-01-25 发布日期:2024-01-16
通讯作者: **（E-mail：nanali@tricaas.com，lili9885@126.com）
作者简介:
^*共同第一作者
基金资助:
国家自然科学基金项目(31700615); 国家自然科学基金项目(32172633); 中国农业科学院农业科技创新工程项目(CAAS-ASTIP-2021-TRICAAS)

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 Published:2024-01-25 Online:2024-01-16

摘要/Abstract

摘要：

克隆获得茶树（Camellia sinensis）镁离子螯合酶H亚基基因CsChlH完整开放阅读框序列（NCBI登录号：ON256194），对应编码1 382个氨基酸残基。系统进化树分析显示，茶树CsChlH与猕猴桃AcChlH的亲缘关系密切。启动子克隆及序列分析显示，CsChlH可能响应光照、干旱、热激、低温、ABA和乙烯等多种信号，且可能调控叶绿素、类黄酮、糖等代谢以及叶绿体、叶肉、保卫细胞等器官发育。CsChlH蛋白定位于叶绿体中。茶树不同组织表达分析显示，CsChlH在富含叶绿素的源组织中表达量高，而在缺乏叶绿素的库组织中表达量低。CsChlH响应长时程弱光、低温、干旱而表达下调，短时程ABA、乙烯以及长时程高温信号能诱导其表达上调。研究结果揭示了CsChlH在茶树叶绿素代谢和胁迫响应中的重要作用。

关键词: 茶树, CsChlH, 启动子, 基因表达, 脱落酸, GUS酶活

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

叶玙璠, 王誉洁, 傅前媛, 王璐, 郝心愿, 丁长庆, 王新超, 曹红利, 李娜娜. 茶树镁离子螯合酶H亚基基因CsChlH的克隆及其表达分析[J]. 园艺学报, 2024, 51(1): 91-102.

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.

图/表 7

表1 茶树CsChlH克隆与表达分析所用引物

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

图1 茶树CsChlH开放阅读框扩增（A）、编码蛋白质序列（B）及基因结构（C）红色下划线区域是蛋白质保守结构域。

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.

图2 茶树CsChlH启动子PCR扩增（A）及测序序列（B）

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

图3 茶树CsChlH蛋白的亚细胞定位 A和E：GFP绿色荧光信号；B：细胞核RFP红色荧光信号；F：叶绿素自发荧光；C和G：明场；D和H：信号融合。

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.

图4 茶树CsChlH的组织特异性表达模式不同小写字母表示具有显著性差异（P < 0.05）。下同。

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

图5 茶树CsChlH在遮荫、植物生长调节剂、温度和干旱处理下的表达水平 * 表示不同处理间差异显著（P < 0.05）。

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）.

图6 注射prCsChlH::GUS烟草经干旱处理后的GUS染色及酶活性检测

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

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茶树镁离子螯合酶H亚基基因CsChlH的克隆及其表达分析

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

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茶树镁离子螯合酶H亚基基因CsChlH的克隆及其表达分析

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

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