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茶叶科学 ›› 2019, Vol. 39 ›› Issue (3): 280-288.doi: 10.13305/j.cnki.jts.2019.03.005

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茶树CsLHTs亚家族基因的克隆与表达分析

郭玲玲,张芬,张亚真,成浩,韦康,阮丽,吴立赟,王丽鸳*   

  1. 中国农业科学院茶叶研究所/国家茶树改良中心,浙江 杭州310008
  • 收稿日期:2019-01-24 修回日期:2019-03-15 出版日期:2019-06-15 发布日期:2019-06-15
  • 通讯作者: 郭玲玲,E-mail:guoxiaoling7125@163.com
  • 作者简介:郭玲玲,女,硕士研究生,主要从事茶树遗传育种方面的研究。*通信作者:wangly@tricaas.com
  • 基金资助:
    国家自然科学基金(31570695)、中央级科研院所基本科研业务费专项(1610212018004)、国家现代农业产业技术体系建设专项资金项目(CARS-19)、浙江省农业新品种选育重大专项(2016C02053)

Molecular Cloning and Expression Analysis of CsLHTs Gene Subfamily in Tea Plants (Camellia sinensis)

GUO Lingling, ZHANG Fen, ZHANG Yazhen, CHENG Hao, WEI Kang, RUAN Li, WU Liyun, WANG Liyuan*   

  1. Tea Research Institute,the Chinese Academy of Agricultural Sciences, National Center for Tea Improvement, Hangzhou 310008, China
  • Received:2019-01-24 Revised:2019-03-15 Online:2019-06-15 Published:2019-06-15
  • Contact: GUO Lingling,E-mail:guoxiaoling7125@163.com

摘要: 氨基酸是茶叶的重要品质成分和氮素贮存形式,因此开展茶树体内氨基酸转运蛋白的研究尤为重要。本研究从茶树转录组中筛选得到5条茶树LHTs(Lysine histidine transporters,赖氨酸/组氨酸转运蛋白)家族基因序列,并从龙井43中成功克隆获得4个茶树LHTs基因,分别命名为CsLHT1CsLHT6CsLHT8.1CsLHT8.2。对该亚家族编码的氨基酸序列的理化性质和功能结构进行预测,结果显示CsLHTs亚家族含有9~11个跨膜区;且含有与氨基酸转运相关的结构域。为了明确在不同茶树品种和氮素水平间CsLHTs基因的表达差异,本研究选用3个茶树品种的扦插苗为试验材料,水培条件下氮饥饿两周后,分别用0.2、2、10 mmolL-1的NH4NO3进行处理。利用qRT-PCR分析了CsLHTs在不同组织间的表达情况,结果表明4个基因在茶树营养组织中均有表达。经氮素处理后,CsLHTs基因在3个氮素水平和3个品种中呈现出不同的变化规律。CsLHT1CsLHT6CsLHT8.2在品种间的表达差异程度高于不同氮素水平间的基因表达差异。CsLHT8.1对氮素处理有明显的响应,尤其经0.2 mmolL-1和10 mmolL-1的NH4NO3处理72 h后,在氮高效品种中茶302根中呈上调表达,表明CsLHT8.1可能参与氨基酸由根部向地上部的转运过程。

关键词: 茶树, LHTs, 氮素, 基因克隆与表达

Abstract: Amino acids are important quality components and forms of nitrogen storage in tea plants (Camellia sinensis). Therefore, it is of great significance to study the function of amino acid transporters. In present study, five LHTs (Lysine histidine transporters, CsLHTs) were found from transcriptomes of tea plants. Among them, four CsLHTs were successfully cloned from the cultivar ‘Longjing 43’ by RT-PCR, and named CsLHT1, CsLHT6, CsLHT8.1 and CsLHT8.2, respectively. The prediction results from amino acid sequences showed that each of CsLHT contained 9-11 transmembrane domains and one domain of amino acid transporter. To investigate the expression patterns of these CsLHT genes under different nitrogen levels, cutting seedlings of three tea cultivars were fed with NH4NO3 of three levels (0.2 mmolL-1, 2 mmolL-1 and 10 mmolL-1) after two weeks of nitrogen starvation. The qRT-PCR results showed that four CsLHTs were expressed in all vegetative tissues, and exhibited diverse expression patterns. For the genes of CsLHT1, CsLHT6 and CsLHT8.2, the gene expression were affected more highly by tea cultivars rather than by nitrogen levels. However, the gene expression of CsLHT8.1 changed greatly by nitrogen treatments. Especially, after 72 h treatments of 0.2 mmolL-1 and 10 mmolL-1 NH4NO3, the CsLHT8.1 expressions were significantly up-regulated in the root of ‘Zhongcha 302’, which was identified as a high nitrogen efficient cultivar. These results implied that CsLHT8.1 might participate in the transport of amino acids from root to aerial parts in tea plants.

Key words: Camellia sinensis, Lysine histidine transporters (LHTs), nitrogen, gene cloning and expression

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