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作物学报 ›› 2024, Vol. 50 ›› Issue (3): 656-668.doi: 10.3724/SP.J.1006.2024.34069

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

茶树CsMCC1和CsMCC2基因的克隆及表达特征性分析

代洪苇(), 刘洁强, 张丽, 童华荣(), 袁连玉()   

  1. 西南大学食品科学学院 / 川渝共建特色食品重庆市重点实验室, 重庆 400715
  • 收稿日期:2023-04-07 接受日期:2023-09-13 出版日期:2024-03-12 网络出版日期:2023-09-28
  • 通讯作者: *袁连玉, E-mail: yuanlianyu88@163.com; 童华荣, E-mail: huart@swu.edu.cn
  • 作者简介:E-mail: hongwdai@163.com
  • 基金资助:
    重庆市技术创新与应用发展专项重点项目(CSTB2022TIAD-CUX0021);重庆市农业农村委重庆市现代山地特色高效农业茶产业技术体系项目(2022-8)

Cloning and relative expression pattern analysis of CsMCC1 and CsMCC2 in tea plant (Camellia sinensis)

DAI Hong-Wei(), LIU Jie-Qiang, ZHANG Li, TONG Hua-Rong(), YUAN Lian-Yu()   

  1. College of Food Science, Southwest University / Chongqing Key Laboratory of Specialty Food Co-built by Sichuan and Chongqing, Chongqing 400715, China
  • Received:2023-04-07 Accepted:2023-09-13 Published:2024-03-12 Published online:2023-09-28
  • Contact: *E-mail: yuanlianyu88@163.com; E-mail: huart@swu.edu.cn
  • Supported by:
    Chongqing Technology Innovation and Application Demonstration Project(CSTB2022TIAD-CUX0021);Chongqing Agriculture and Rural Affairs Commission Chongqing Modern Mountain Characteristic Efficient Agriculture Tea Industry Technology System(2022-8)

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摘要：

组蛋白乙酰化修饰由组蛋白乙酰化酶(histone acetyltransferases, HATs)和脱乙酰化酶(histone deacetylase, HDACs)共同催化完成, 是重要的表观遗传调控方式之一, 在植物生长发育、逆境胁迫响应和激素响应的调控过程中均具有重要意义, 但对茶树(Camellia sinensis)组蛋白乙酰化修饰的研究还比较少。本研究从‘福鼎大白茶’茶树中克隆获得了2个HATs家族的MCC (MEIOTIC CONTROL OF CROSSOVERS)基因: CsMCC1和CsMCC2, 通过生物信息学分析、实时荧光定量PCR技术 (Real-time quantitative PCR, qRT-PCR)和亚细胞定位试验对其功能进行解析。结果显示, CsMCC1和CsMCC2基因分别位于茶树1号和7号染色体, 分别编码257 aa和269 aa, 均属于碱性不稳定亲水性蛋白, 与拟南芥AtMCC1具有高度相似的基因结构和蛋白空间结构。系统进化树和保守结构域分析表明, CsMCC蛋白与葡萄、番茄的同源蛋白有较近的亲缘关系, MCC蛋白序列高度保守, 均含有GNAT保守结构, 属于HAT蛋白的GNAT (GCN5-related N-terminal acetyltransferases)亚家族。拟南芥原生质体亚细胞定位结果显示, CsMCC1和CsMCC2蛋白均定位于细胞质膜。启动子分析显示, 在CsMCC1和CsMCC2基因启动子中包含多个与胁迫、光和植物激素调节响应相关的元件。转录组数据和表达分析发现, CsMCC1基因在叶、花和根发育早期的表达量明显高于后期; CsMCC2基因在茶树根部有最高表达, 并在根部发育的较长阶段均有较高水平的表达量; 2个CsMCC基因均能够被多种非生物胁迫(干旱、盐和冷)和外源激素(MeJA、GA3和IAA)诱导表达。蛋白相互作用预测分析显示, CsMCC蛋白与多个乙酰化转移酶相关蛋白具有关联性。综上所述, CsMCC1和CsMCC2基因具有通过组蛋白乙酰化修饰作用广泛参与茶树的生长发育和环境响应过程的潜能, 可为进一步研究CsMCC基因在茶树中的功能提供理论参考。

关键词: 茶树, 组蛋白乙酰化, CsMCC基因, 表达分析

Abstract:

Histone acetylation is an essential type of epigenetic modifications, which is mainly catalyzed by histone acetylases (HATs) and deacetylases (HDACs) and plays a crucial role in plant growth, stress response, and hormone regulation. However, little research information is available about tea plants histone acetylases. We cloned two MCC (MEIOTIC CONTROL OF CROSSOVERS) genes (CsMCC1 and CsMCC2) of HATs family from the ‘Fuding Dabaicha’ tea plant. Meanwhile, the function of these two CsMCC genes was analyzed by bioinformatics methods, qRT-PCR, and subcellular localization. Results showed that the CsMCC1 and CsMCC2 genes were located on chromosomes 1 and 7, encoding alkaline unstable hydrophilic proteins of 257 and 269 amino acids, respectively. The CsMCC genes and protein structure of tea plant were similar to those of the Arabidopsis AtMCC1 gene. The phylogenetic tree and conserved structural domain analysis showed that the MCC protein belonged to the GNAT (GCN5-related N-terminal acetyltransferases) subfamily of HATs proteins, and contained GNAT conserved structures. The evolutionary relationship of CsMCC proteins was closely related to the MCC members in grapes and tomatoes with the highly conserved protein sequences. The subcellular localization in Arabidopsis protoplasts revealed that the CsMCC1 and CsMCC2 proteins were localized on the cytoplasmic membrane. And the promoters of CsMCC1 and CsMCC2 genes contained a number of elements involved in responses to stress, light, and phytohormones. According to transcription data and expression analysis, the relative expression level of CsMCC1 gene was significantly higher in the younger stages of leaf, flower, and root development than the older stages, and CsMCC2 gene was higher in root than other tissues and lasted for a longer time during root development period. The CsMCC1 and CsMCC2 could be regulated by various abiotic stresses (drought, salt, and cold) and exogenous hormones (MeJA, GA3, and IAA). In addition, CsMCC proteins could interact with acetyltransferase-related proteins. Hence, CsMCC genes might play roles in tea plant growth and development, and the response to environment through histone acetylation modification. This study explored the basic features and functions of CsMCC, providing the useful theoretical reference for further research on the functions of CsMCC genes in tea plant.

Key words: tea plant, histone acetylation, CsMCC genes, relative expression analysis

表1

PCR扩增和基因表达分析的引物"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 用途
Primer function
CsMCC1 ATGCCATATTCTTCAATGGCA & TCAACTCTGCTCTTTATAGGG 基因克隆
Gene cloning
CsMCC2 ATGCCAATTTTTTGCTGGGGA & CTACACACATTGAAACCCGGT
CsMCC1-Q GGCGTATACGACCCTCTGATT & TCAACTGCTCCCCAGGATACA 基因表达分析
Gene relative expression level
CsMCC2-Q AGGTGTCCTCCATCCAACCAT & CAGTCAACAGCTCCCCAAGAT
CsACTIN CCAGAAAGATGCTTATGTAG & AGATCTTTTCCATGTCATCC
CsMCC1-p AAGTCCGGAGCTAGCTCTAGatgccatattcttcaatggcagacttgaaagg & AGCGGCCGCTGTACAGGATCaaactcggtaccatcagtcacaggg 亚细胞定位
Subcellular localization
CsMCC2-p AAGTCCGGAGCTAGCTCTAGatgccaattttttgctggggattaatgcaacc & AGCGGCCGCTGTACAGGATCcacacattgaaacccggtaccttgag

图1

茶树CsMCC基因的克隆和理化性质分析 (A): CsMCC基因的凝胶电泳图; (B): CsMCC基因染色体定位; (C): CsMCC和AtMCC1基因结构分析; (D): CsMCC1蛋白跨膜结构预测; (E): CsMCC蛋白亲疏水性预测, 数值小于0表示亲水, 大于0表示疏水。"

表2

茶树CsMCC1和CsMCC2 蛋白的理化性质"

基因
Gene		 基因位置
Gene location		 编码长度
Encoding length (bp)		 蛋白分子质量
Protein molecular weight (kD)		 等电点
Isoelectric point		 跨膜结构
Transmembrane structures		 不稳定指数
Instability index
CsMCC1 (TEA029044) Scaffold1047: 74780-82234 774 29.34 9.44 1 46.50
CsMCC2 (TEA011651) Scaffold7556: 73159-84022 810 30.69 8.22 0 46.13

图2

HAT蛋白家族(A)和植物MCC蛋白(B)的进化树分析 构建进化树的MCC同源蛋白来自茶树(Camellia sinensis, CsMCC1/2), 拟南芥(Arabidopsis thaliana, AtMCC1), 番茄(Solanum lycopersicum, Solyc03T002163), 玉米(Zea mays, Zm00001d017691), 水稻(Oryza sativa, LOC_Os02g46700), 葡萄(Vitis vinifera, VIT_216s0039g01810), 山核桃(Carya illinoinensis Pawnee, CiPaw.05G216100), 栗树(Castanea dentata, Caden.12G113400), 黄瓜(Cucumis sativus, Cucsa.318740), 棉花(Gossypium darwinii, Godar.D08G277700, Godar.A08G263100), 芥菜(Capsella rubella, Carub.0003s0211), 芝麻菜(Eruca vesicaria, Eruve.2849s0002), 板蓝根(Isatis tinctoria, Isati.1576s0019), 柑橘(Citrus clementina, Ciclev10021817m), 毛果杨(Populus trichocarpa, Potri.013G079900)。"

图3

MCC蛋白序列特征分析 (A): CsMCC和AtMCC1蛋白的保守基序分析; (B): MCC蛋白的保守结构域示意图; (C): 植物MCC蛋白的多序列比对。"

图4

茶树CsMCC1和CsMCC2蛋白的空间结构分析 (A): 蛋白二级结构; (B): 蛋白三级结构。"

图5

CsMCC在拟南芥原生质体中的亚细胞定位"

图6

CsMCC基因启动子区域的顺式作用元件分析 (A): 启动子元件分布, 不同颜色对应下方图的不同元件; (B): 以热图形式展示启动子元件数量, 灰色方格表示不含有该元件。"

图7

茶树CsMCC基因的时空特异性表达分析 (A): CsMCC1和CsMCC2基因在茶树不同组织的表达分析; (B)~(E): 分别为CsMCC1和CsMCC2基因在茶树芽、叶片、花和根的不同发育时期的表达分析。"

图8

茶树CsMCC基因在非生物胁迫和激素处理下的表达分析 (A): 以25% PEG模拟茶树干旱环境, CK_P、PEG_24 h/48 h/72 h分别表示用PEG处理0 h、24 h、48 h和72 h; (B): 以11.7 g L-1 NaCl模拟茶树的盐胁迫, CK_N、NaCl_24 h/48 h/72 h分别表示用NaCl处理0 h、24 h、48 h和72 h; (C): 冷驯化处理, CK_C为对照, CA1为冷驯化, CA2为去驯化; (D): CK_M、MeJA_12 h/24 h/48 h分别表示用MeJA处理0 h、12 h、24 h和48 h; (E): CK_G、GA3_24 h/48 h分别表示用100 mg L-1 GA3处理0 h、24 h和48 h; (F): CK_I、IAA_2 h/48 h分别表示用50 mg L-1 IAA处理0 h、24 h和48 h。数据经标准归一化处理后, 在TBtools中绘制热图, 红色表示上调表达, 蓝色表示下调表达, 颜色越深表示表达量变化越大。"

图9

CsMCC蛋白的相互作用网络分析"

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