Journal of Nuclear Agricultural Sciences ›› 2021, Vol. 35 ›› Issue (6): 1281-1290.DOI: 10.11869/j.issn.100-8551.2021.06.1281

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

Cloning and Expression Analysis of Three MAPKKK Genes in Postharvest Processing of Tea Leaves

SHEN Chaoru, YANG Runmei, YUE Chuan, CAO Hongli*   

  1. College of Horticulture, Fujian Agriculture and Forestry University/Key Laboratory of Tea Science in Universities of Fujian Province, Fuzhou, Fujian 350002
  • Received:2020-10-09 Online:2021-06-10 Published:2021-04-16

茶树3个MAPKKK基因的克隆及其在采后加工中的表达

沈潮如, 杨润梅, 岳川, 曹红利*   

  1. 福建农林大学园艺学院/茶学福建省高校重点实验室,福建 福州 350002
  • 通讯作者: *曹红利,女,讲师,主要从事茶树逆境分子生物学与品质调控研究。E-mail:lili9885@126.com
  • 作者简介:沈潮如,女,主要从事茶树资源与品质调控研究。E-mail:2751987133@qq.com
  • 基金资助:
    国家自然科学基金资助项目(31800587、 31600555),国家茶叶产业技术体系项目(CARS-19),福建农林大学茶产业链科技创新与服务体系建设项目(K1520005A01)

Abstract: Mitogen-activated protein kinase (MAPK) cascades play essential roles in plant response to biotic and abiotic stresses. In order to study the function of MAPKKK genes during tea postharvest processing, the full length cDNA of three MAPKKK genes were cloned from tea cultivar ‘Fudingdabaicha’cultivar, and named as CsMAPKKK18, CsMAPKKK18-like and CsMAPKKK-NPK1, respectively. Bioinformatic analyses showed that the coding sequence of CsMAPKKK18, CsMAPKKK18-like and CsMAPKKK-NPK1 are 1 005 bp, 1 209 bp and 1 077 bp in length, and encode 334, 402 and 358 amino acids putatively located on the vacuole, mitochondria and nucleus respectively. Conserve domain analysis showed that three CsMAPKKK belonged to MEKK family with conservative region of G(T/S)PX(W/Y/F)MAPEV. Phylogenetic tree analysis showed that CsMAPKKK18 and CsMAPKKK18-like have the closest relationship to homologies in Solanum lycopersicum and Capsicum annuum while MAPKKK-NPK1 corresponding to Ricinus communis and Vitis vinifera. qRT-PCR analysis showed that the CsMAPKKK genes were highly expressed in roots. In the processing of white tea withering, CsMAPKKKs were up-regulated significantly, and particularly the expression level of CsMAPKKK18 raised up to 65-fold. In the green-making procedure of Oolong tea, the transcription level of CsMAPKKK18 increased to 26-fold. CsMAPKKK18-like was significantly up-regulated at the third tumbling procedure and down-regulated at the solar withering, second tumbling and before fixation procedures. CsMAPKKK-NPK1 was dramatically down-regulated at the solar withering procedure and up-regulated at the first, second and third tumbling procedures. These results demonstrated that CsMAPKKKs might be involved to the withering processing of white tea and green-making procedures of oolong tea, and provided a foundation for further studies on the quality formation mechanism of tea processing.

Key words: Camellia sinensis, mitogen-activated protein kinase kinase kinase(MAPKKK), white tea withering, Oolong tea rotation, gene expression

摘要: 丝裂原活化蛋白激酶(MAPK)级联在植物应对生物和非生物胁迫过程中发挥重要作用。为了研究MAPKKK基因在应答茶树采后加工中的功能,本研究以福鼎大白茶品种为材料,克隆了3个MAPKKK基因,分别命名为CsMAPKKK18、CsMAPKKK18-likeCsMAPKKK-NPK1。生物信息学分析结果显示,CsMAPKKK18包含1 005 bp开放阅读框,编码334个氨基酸,预测定位于液泡中;CsMAPKKK18-like包含1 209 bp开放阅读框,编码402个氨基酸,预测定位于线粒体中;CsMAPKKK-NPK1包含1 077 bp开放阅读框,编码358个氨基酸,预测定位于细胞核中。3个MAPKKK蛋白均具有MEKK特征性保守区域G(T/S)PX(W/Y/F)MAPEV,属于MEKK亚家族。进化树分析显示,CsMAPKKK18与CsMAPKKK18-like都与番茄、甜椒亲缘关系较近,MAPKKK-NPK1与蓖麻和葡萄关系最近。荧光定量PCR检测结果显示,3个CsMAPKKK基因都在根中表达量较高。在白茶萎凋过程中,3个MAPKKK基因都显著上调表达,其中CsMAPKKK18表达量最高上调达65倍。在乌龙茶做青过程中,CsMAPKKK18表达量显著上调最大为26倍;CsMAPKKK18-like在三摇显著上调,但在晒青、二摇和杀青前显著下调表达;CsMAPKKK-NPK1在晒青过程中显著下调,在一摇、二摇和三摇都显著上调。研究结果表明CsMAPKKKs基因在白茶萎凋过程及乌龙茶做青过程中可能发挥作用,这为后续研究茶叶加工过程中品质形成的分子调控机理提供参考。

关键词: 茶树, 丝裂原活化蛋白激酶激酶激酶(MAPK), 白茶萎凋, 乌龙茶做青, 基因表达