茶树叶片黄化变异相关的CAB基因家族鉴定及关键基因挖掘

钟思彤; 张亚真; 游小妹; 陈志辉; 孔祥瑞; 林郑和; 伍慧妮; 金珊; 陈常颂

doi:10.13305/j.cnki.jts.2024.02.007

茶叶科学 >

2024 , Vol. 44 >Issue 2: 175 - 192

DOI: https://doi.org/10.13305/j.cnki.jts.2024.02.007

研究报告

茶树叶片黄化变异相关的CAB基因家族鉴定及关键基因挖掘

钟思彤 ,
张亚真 ,
游小妹 ,
陈志辉 ,
孔祥瑞 ,
林郑和 ,
伍慧妮 ,
金珊 ,
陈常颂

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1.福建农林大学园艺学院,福建福州 350002;
2.福建省农业科学院茶叶研究所/国家茶树改良中心福建分中心,福建福州 350012

钟思彤,女,硕士研究生,主要从事茶树遗传育种与分子生物学研究。

收稿日期: 2024-01-15

修回日期: 2024-03-11

网络出版日期: 2024-04-30

基金资助

福建省属公益类科研院所基本科研专项（2022R1029002、2021R1029003、2021R1029007）、国家现代农业产业技术体系（CARS-19）、农业高质量发展超越“5511”协同创新工程（XTCXGC2021004）、福建省农业科学院科技创新团队（CXTD2021006-1）

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Identification of CAB Gene Family and Excavation of Key Genes Related to Leaf Yellowing Variationin Tea Plants (Camellia sinensis)

ZHONG Sitong ,
ZHANG Yazhen ,
YOU Xiaomei ,
CHEN Zhihui ,
KONG Xiangrui ,
LIN Zhenghe ,
WU Huini ,
JIN Shan ,
CHEN Changsong

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1. College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
2. Tea Research Institute, Fujian Academy of Agricultural Sciences/Fujian Branch, National Center for Tea Improvement, Fuzhou 350012, China

Received date: 2024-01-15

Revised date: 2024-03-11

Online published: 2024-04-30

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摘要

捕光叶绿素a/b结合蛋白（Light-harvesting chlorophyll a/b binding protein,CAB）基因家族成员在植物叶片黄化变异中具有重要作用。本研究利用铁观音（Camellia sinensis Tieguanyin）基因组数据对CAB基因家族进行了筛选,并进行了生物信息学和表达模式分析;进一步以不同黄化、绿叶茶树品种（系）为材料,通过基因克隆和qRT-PCR,分析CABs基因的表达特性,结合叶色参数和叶绿素SPAD值的相关性分析筛选出与茶树黄化变异相关的关键CAB基因。结果表明,共鉴定到25个CAB基因家族成员,其氨基酸长度为167~337个,蛋白分子质量为18.5~37.1 kDa,大部分CAB成员属于稳定性蛋白和疏水性蛋白,并且亚细胞定位预测在叶绿体上;根据进化关系25个CAB家族成员分为13个亚家族,Lhcb1亚族的成员数量最多;启动子分析显示,CAB家族成员启动子中包含大量的光响应元件,还有其他与植物生长发育、激素和逆境胁迫响应相关的元件。从茶树中克隆Lhcb1亚家族成员,通过序列比对筛选出CAB1、CAB6和CAB7基因;表达分析显示,CAB1、CAB6和CAB7基因都具有组织表达特异性,在芽、叶和果实中表达量较高,并能响应多种逆境胁迫。qRT-PCR分析发现,CAB1、CAB6和CAB7基因在黄、绿茶树的叶片中具有一致的表达特性：与正常绿叶相比,黄化叶片中的CABs基因表达均显著下调;通过与叶色参数和叶绿素SPAD值的相关性分析,发现CAB1、CAB6和CAB7表达量与叶色参数a、b、L值以及叶绿素SPAD值均呈极显著相关（P<0.01）,其中CAB1的基因表达量与叶色相关参数和叶绿素SPAD值的相关性最显著;烟草亚细胞定位结果显示,CAB1在细胞核、细胞膜和叶绿体上均有分布。以上研究初步解析了茶树CAB家族成员的基本特征,挖掘出与茶树叶色变异紧密相关CAB基因,后续可作为候选关键基因进行深入研究,以期为茶树叶色变异的分子调控机制的研究提供理论基础。

关键词： 茶树; 捕光叶绿素a/b结合蛋白; 叶色黄化变异; 基因克隆; 表达分析

本文引用格式

钟思彤 , 张亚真 , 游小妹 , 陈志辉 , 孔祥瑞 , 林郑和 , 伍慧妮 , 金珊 , 陈常颂 . 茶树叶片黄化变异相关的CAB基因家族鉴定及关键基因挖掘[J]. 茶叶科学, 2024 , 44(2) : 175 -192 . DOI: 10.13305/j.cnki.jts.2024.02.007

Abstract

Members of the light-harvesting chlorophyll a/b binding protein (CAB) gene family play an important role in plant leaf yellowing variation. In this study, the CAB family members were identified from tea plant ‘Tieguanyin’ genomic data. The bioinformatics and expression patterns were analyzed. Furthermore, the expression patterns of the CABs gene were analyzed by gene cloning and qRT-PCR in tea cultivars with different leaf colors. The key CAB genes related to tea yellowing were screened by correlation analysis of leaf color parameters and chlorophyll SPAD values. The results show that 25 members of the CAB gene family were identified, their amino acid length ranged from 167-337 and the protein molecular weight ranged from 18.5-37.1 kDa. Most CAB members were stable and hydrophobic proteins, and distributed in chloroplast by the subcellular localization prediction. According to the evolutionary relationship, CAB family members are divided into 13 subfamilies, and the Lhcb1 subfamily has the most members. Cis-acting element analysis of promoter shows that CAB family members have a lot of light-responsive elements, as well as other elements related to growth and development, hormone response, and adversity stress. The members of Lhcb1 subfamily were cloned from tea plants, CAB1, CAB6, and CAB7 genes were screened by sequence alignment. The expression analysis shows that CAB1, CAB6, and CAB7 genes had tissue expression characteristics with higher expression levels in buds, leaves and fruits, and could respond to various stresses. Finally, the qRT-PCR indicates that the expressions of CAB1, CAB6, and CAB7 genes were consistent in the yellow and green leaves. Compared with green leaves, the expression of CAB genes in yellow leaves were significantly down-regulated. The correlation analysis of gene expressions and related leaf color parameters shows that the gene expressions of CAB1, CAB6, and CAB7 were significantly correlated with leaf color parameters a, b, L, and chlorophyll SPAD values (P<0.01). Among them, the expression of CAB1 shows the highest correlation coefficient. The subcellular localization analysis shows that CAB1 was distributed in the nucleus, cytoplasm, and chloroplasts. The studies analyzed the basic characteristics of CAB family members in tea plants and the key genes related to tea color variation were identified, which provided a theoretical basis for the molecular regulation mechanism of tea color variation.

Key words： Camellia sinensis; CAB; yellowing variation; gene cloning; expression analysis

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