茶树CsCRD1基因的克隆及其对叶绿素生物合成的影响

doi:10.13560/j.cnki.biotech.bull.1985.2025-1048

摘要/Abstract

摘要：

目的 Mg-原卟啉Ⅸ单甲基酯环化酶（Mg-protoporphyrin Ⅸ monomethyl ester cyclase, MgPEC）是叶绿素生物合成途径中的关键酶，探究编码该酶的CsCRD1基因对茶树叶绿素生物合成的影响，有助于解析茶树叶色形成的分子机制，为选育叶色变异的茶树品种提供科学的理论依据。方法利用PCR技术克隆CsCRD1基因，对其进行生物信息学、系统发育、表达模式、亚细胞定位分析，并采用反义寡核苷酸沉默技术探究CsCRD1的功能。结果茶树CsCRD1的CDS长度为1 215 bp，编码404个氨基酸，蛋白分子量为46.87 kD，pI为8.56，定位于茶树第10号染色体；亚细胞定位结果显示CsCRD1蛋白定位于烟草叶肉细胞的叶绿体内。CsCRD1蛋白含高度保守的Rubrerythrin结构域，与番茄CRD1亲缘关系最近，保守基序种类和顺序与其他植物CRD1一致，进化上高度保守。启动子元件分析发现，CsCRD1含光响应、激素响应及逆境胁迫响应元件。组织特异性表达分析显示，CsCRD1在茶树嫩叶及成熟叶中的相对表达量最高，花中最低。不同叶色茶树中，绿色叶片中CsCRD1的表达量显著高于白化叶片，且白化叶片返绿过程中该基因表达量会提高。相关性分析表明，CsCRD1的表达水平与茶树叶片叶绿素含量呈正相关。反义寡核苷酸沉默试验结果发现，CsCRD1沉默后叶绿素a、b及总叶绿素含量显著下降，叶绿素合成相关基因CsCHLM、CsDVR、CsPORA表达下调。结论茶树CsCRD1定位于叶绿体，在绿色叶片中高表达，抑制CsCRD1表达会减少叶绿素的生物合成，从而证实其在叶绿素代谢及叶色形成过程中具有关键作用。

关键词: 茶树, CsCRD1, 基因克隆, 生信分析, 表达分析, 亚细胞定位, 叶绿素生物合成, 反义寡核苷酸沉默

Abstract:

Objective Mg-protoporphyrin Ⅸ monomethyl ester cyclase (MgPEC) is a key enzyme in chlorophyll biosynthesis pathway. Exploring the effect of the CsCRD1 gene that encodes this enzyme, on chlorophyll biosynthesis will help elucidate the molecular mechanisms of leaf color formation in tea plants, providing a scientific theoretical basis for the selection and breeding of tea plant varieties with leaf color mutations. Method The CsCRD1 gene was cloned using PCR technology. Bioinformatics, phylogenetic, expression pattern, and subcellular localization analyses were performed. Antisense oligonucleotide silencing technology was used to explore the function of CsCRD1. Result The CDS length of CsCRD1 is 1 215 bp, encoding 404 aa, with a molecular weight of 46.87 kD and the pI of 8.56. CsCRD1 is located on chromosome 10, and subcellular localization results showed that the CsCRD1 protein is located in the chloroplasts of tobacco mesophyll cells. The CsCRD1 protein contains a highly conserved Rubrerythrin domain, and it is most closely related to the tomato CRD1. The conserved motif types and sequence are consistent with other plant CRD1 proteins, indicating strong evolutionary conservation. Promoter element analysis revealed that it contains light-responsive, hormone-responsive, and stress-responsive elements. Tissue-specific expression analysis showed that CsCRD1 is most highly expressed in the young and mature leaves of tea plants, and least in the flowers. In tea plants with different leaf colors, the relative expression of CsCRD1 in green leaves is significantly higher than in albino leaves, and the expression of this gene increases during the re-greening process of the albino leaves. Correlation analysis showed that the expression level of CsCRD1 is positively correlated with chlorophyll content in tea leaves. Results from antisense oligonucleotide silencing experiments showed that silencing CsCRD1 significantly reduces the content of chlorophyll a, b, and total chlorophyll, and downregulates the expressions of chlorophyll biosynthesis-related genes CsCHLM, CsDVR, and CsPORA. Conclusion CsCRD1 is localized in the chloroplasts and highly expressed in green leaves. Silencing CsCRD1 expression reduces chlorophyll biosynthesis, thus confirming its critical role in chlorophyll metabolism and leaf color development.

Key words: tea plant, CsCRD1, gene cloning, bioinformatics analysis, expression analysis, subcellular localization, chlorophyll biosynthesis, silencing of antisense oligonucleotide

张欣, 陈骋, 霍元博, 黄仁丽, 张丽, 张凯, 童华荣, 袁连玉. 茶树CsCRD1基因的克隆及其对叶绿素生物合成的影响[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1048.

ZHANG Xin, CHEN Cheng, HUO Yuan-Bo, HUANG Ren-Li, ZHANG Li, ZHANG Kai, TONG Hua-rong, YUAN Lian-Yu. Cloning of the CsCRD1 in Tea Plants and Its Impact on Chlorophyll Biosynthesis[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1048.

图/表 9

图1 茶树CsCRD1基因的克隆和特征分析A：CsCRD1基因的琼脂糖凝胶电泳图；B：CsCRD1基因染色体定位；C：CsCRD1基因结构分析

Fig. 1 Cloning and characterization analysis of CsCRD1 gene in tea plantA: Agarose gel electrophoresis image of the CsCRD1 gene. B: Chromosomal localization of CsCRD1 gene. C: Gene structural analysis of CsCRD1

图2 植物CRD1蛋白的系统进化树和序列特征分析A：植物CRD1蛋白的系统进化树；构建系统进化树的CRD1同源蛋白来自茶树（Camellia sinensis， CsCRD1），拟南芥（Arabidopsis thaliana， AtCRD1），番茄（Solanum lycopersicum， Solyc10T002193），玉米（Zea mays， Zm00001d040463），水稻（Oryza sativa， LOC_Os02g46700），棉花（Gossypium darwinii， Godar.D01G235000），芥菜（Brassica juncea， Carub.0005s2368），芝麻菜（Eruca vesicaria， Eruve.0383s0052），毛果杨（Populus trichocarpa， Potri.006G027300），葡萄（Vitis vinifera， VIT_208s0040g00390），黄瓜（Cucumis sativus， Cucsa.059080），甜橙（Citrus sinensis， orange1.1g014877m），山核桃（Carya illinoinensis Pawnee， CiPaw.09G162600），柑橘（Citrus clementina， Ciclev10011842m），芒草（Miscanthus， Misin06G112500）；B：植物CRD1蛋白的保守基序；C：植物CRD1蛋白的保守结构域示意图；D：植物CRD1蛋白的氨基酸多序列比对；蓝黑色：氨基酸一致性为100%；粉红色：氨基酸一致性为75%；蓝色：氨基酸一致性为50%。▬：植物CRD1蛋白的典型保守结构域Rubreythin

Fig. 2 Analysis of phylogenetic tree of CRD1 proteins and its sequence characteristics in plantA: Phylogenetic tree of CRD1 proteins in plant. The CRD1 homologous proteins used to construct the phylogenetic tree come from tea plant (Camellia sinensis, CsCRD1), Arabidopsis (Arabidopsis thaliana, AtCRD1), tomato (Solanum lycopersicum, Solyc10T002193), corn (Zea mays, Zm00001d040463), rice (Oryza sativa, LOC_Os02g46700), cotton (Gossypium darwinii, Godar.D01G235000), mustard (Brassica juncea, Carub.0005s2368), arugula (Eruca vesicaria, Eruve.0383s0052), poplar (Populus trichocarpa, Potri.006G027300), grape (Vitis vinifera, VIT_208s0040g00390), cucumber (Cucumis sativus, Cucsa.059080), sweet orange (Citrus sinensis, orange1.1g014877m), pecan (Carya illinoinensis Pawnee, CiPaw.09G162600), clementine (Citrus clementina, Ciclev10011842m), and Miscanthus (Miscanthus, Misin06G112500). B: Conserved motifs of CRD1 proteins in plant. C: The schematic representation for conserved structural domains of CRD1 proteins in plant. D: Amino acid multiple sequence alignment of CRD1 proteins in plant. Blue-black: Amino acid identity is 100%. Pink: Amino acid identity is 75%; blue: amino acid identity is 50%. ▬ indicates predicted the typical conserved domain Rubreythin of CRD1 protein in plants

图3 茶树CsCRD1基因启动子顺式作用元件分析A：启动子顺式作用元件的分布；B：启动子顺式作用元件的数量

Fig. 3 Analysis of cis-acting elements in the promoters of gene CsCRD1 in tea plantA: Distribution of cis-acting elements in the promoter. B: Quantity of cis-acting elements in the promoter

图4 茶树CsCRD1蛋白的亚细胞定位GFP：绿色荧光信号；Chlorophyll fluorescence：叶绿体自发荧光；Bright：明场；Merged：信号融合

Fig. 4 Subcellular localization of CsCRD1 protein in tea plantGFP: Green fluorescent signal. Chlorophyll fluorescence: chloroplast autofluorescence. Bright: Bright field. Merged: Signal merge

图5 茶树CsCRD1基因的组织特异性表达分析A：CsCRD1基因在茶树不同组织中的相对表达水平；B：CsCRD1基因在‘福鼎大白茶’和‘黄金叶’茶树不同叶发育时期的相对表达水平。不同小写字母表示在P<0.05水平差异显著（单因素方差分析结合Tukey事后检验）。下同

Fig. 5 Analysis of tissue-specific expression of CsCRD1 gene in tea plantA: Relative expression of the CsCRD1 gene in different tissues of the tea plant. B: Relative expression of the CsCRD1 gene in different leaf development stages of leaves from ‘Fudingdabaicha’ and ‘Huangjinye’. Different lowercase letters indicate significant differences at P<0.05 level (One-way ANOVA with Tukey’s post hoc test). The same below

图6 茶树CsCRD1基因在叶色变化过程中的特异性表达分析A：CsCRD1基因的相对表达水平；B：叶绿素a含量；C：叶绿素b含量；D：叶绿素总量；E：叶绿素总量与CsCRD1基因相对表达水平的相关性分析

Fig. 6 Expression analysis of the CsCRD1 gene during the leaf color change process in tea plantsA: Relative expression of the CsCRD1 gene. B: Chlorophyll a content. C: Chlorophyll b content. D: Total chlorophyll content. E: Correlation analysis between total chlorophyll content and the relative expression of the CsCRD1 gene

图7 茶树CsCRD1蛋白的相互作用网络分析

Fig. 7 Interacting network analysis of CsCRD protein 1 in tea plant

图8 茶树CsCRD1基因沉默对叶绿素合成响应的分析A：CsCRD1基因AsODN沉默示意图；B：AsODNs-CsCRD1沉默后CsCRD1基因的相对表达水平；C‒E：CsCRD1基因沉默后叶绿素含量的变化；F‒H：CsCRD1基因沉默后叶绿素生物合成途径相关基因的相对表达水平。*、**和***分别表示P<0.05、P<0.01和P<0.001的显著性水平（单因素方差分析结合Tukey事后检验）

Fig. 8 Analysis of the impact of CsCRD1 gene silencing on chlorophyll biosynthesis in tea plantsA: Schematic diagram of CsCRD1 gene AsODN silencing. B: Relative expression of the CsCRD1 gene after AsODNs-CsCRD1 silencing. C‒E: Changes in chlorophyll content after CsCRD1 gene silencing. F‒H: Relative expressions of chlorophyll biosynthesis-related genes after CsCRD1 gene silencing. *, ** and *** indicate the significances of P<0.05, P<0.01, and P<0.001, respectively (one-way ANOVA with Tukey’s post hoc test)

图9 茶树CsCRD1基因对叶绿素生物合成的影响

Fig. 9 Effects of CsCRD1 gene on chlorophyll biosynthesis in tea plants

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