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茶树钙依赖性蛋白激酶基因CsCDPK17的鉴定及表达分析

  • LEI Lei ,
  • WANG Lu ,
  • YAO Lina ,
  • HAO Xinyuan ,
  • ZENG Jianming ,
  • DING Changqing* ,
  • WANG Xinchao* ,
  • YANG Yajun
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  • 中国农业科学院茶叶研究所/国家茶树改良中心/农业农村部茶树生物学与资源利用重点实验室,浙江 杭州 310008
雷蕾,硕士研究生,主要从事茶树遗传育种与抗逆机理研究。*通信作者:chqding@tricaas.com,xcw575@tricaas.com

收稿日期: 2018-11-29

  修回日期: 2018-12-29

  网络出版日期: 2019-06-15

基金资助

国家自然科学基金(31770735)、现代农业(茶叶)产业技术体系(CARS-19)、浙江省农业新品种选育重大科技专项(2016C02053-4)

Identification and Expression Analysis of Calcium-dependent Protein Kinase CsCDPK17 in Tea Plant (Camellia sinensis)

  • 雷蕾,王璐,姚丽娜,郝心愿,曾建明,丁长庆*,王新超*,杨亚军
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  • Tea Research Institute of the Chinese Academy of Agricultural Sciences/National Center for Tea Improvement/Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture and Rural Affairs, Hangzhou 310008, China

Received date: 2018-11-29

  Revised date: 2018-12-29

  Online published: 2019-06-15

摘要

钙依赖型蛋白激酶(Calcium-dependent protein kinases,CDPKs或CPKs)是植物细胞中重要的钙离子信号受体,普遍参与了植物生长发育和胁迫响应等调控过程。本研究从茶树龙井43中克隆到1条CDPK基因,经测序验证该序列包含1 611 bp的完整ORF,编码536个氨基酸。结合序列比对和进化分析发现该蛋白N-末端具有豆蔻酰化位点,具备钙依赖性蛋白激酶的保守结构域,并且与拟南芥AtCDPK17的同源关系最近,因此命名为CsCDPK17(Genbank登录号为:MK238482)。蛋白质理化特性分析发现其蛋白分子量为59.9 kD,理论等电点pI为5.43,属无跨膜结构域的亲水性蛋白。使用水稻原生质体和烟草叶片两种瞬时表达的方法均证明CsCDPK17是细胞质膜和细胞核双定位蛋白。对克隆到的CsCDPK17上游2 000 bp的启动子区分析发现了多个与基因转录、光、激素(ABA、SA、MeJA等)相关的顺式作用元件。表达分析显示,CsCDPK17在成熟叶和种子中表达水平较高,而在根中的表达水平最低;在龙井43、大面白等4个品种冷驯化过程中,该基因的表达随着冷驯化过程显著上调,随着脱驯化过程下调;同时还发现CsCDPK17能够不同程度地响应低温(最高5.1倍)、干旱(最高2.3倍)、渗透(最高2.4倍)胁迫。本研究的结果表明CsCDPK17在茶树的生长发育和低温、干旱、渗透等非生物胁迫响应的过程中均发挥一定的调控作用。

本文引用格式

LEI Lei , WANG Lu , YAO Lina , HAO Xinyuan , ZENG Jianming , DING Changqing* , WANG Xinchao* , YANG Yajun . 茶树钙依赖性蛋白激酶基因CsCDPK17的鉴定及表达分析[J]. 茶叶科学, 2019 , 39(3) : 267 -279 . DOI: 10.13305/j.cnki.jts.2019.03.004

Abstract

Calcium-dependent protein kinases (CDPKs or CPKs) are important calcium sensors in higher plants, which are extensively involved in plant development and stress responding. In this study, one sequence that contained a complete ORF of 1 611 bp encoding a 568 amino acids protein was cloned from Camellia sinensis cv. Longjing 43. Sequence alignments revealed that this protein was a typical plant CDPK possesses N-terminus myristoylation site and protein kinases domain and showed the highest similarity with Arabidopsis AtCDPK17. Thus, the gene was defined as CsCDPK17 base on further phylogenetic analysis (Genbank accession No. MK238482). Basic protein character analysis shows that CsCDPK17 was a hydrophilic membrane-binding protein with molecularweight of 59.9 kD and PI of 5.43. Further subcellular localization assay using transient CsCDPK17-GFP expression in rice protoplasts and tobacco leaves proved that CsCDPK17 was localized in plasma membrane and nucleus. A series of gene transcription, light and hormone (such as ABA, SA, MeJA, etc) responding related cis-elements were detected in CsCDPK17 2 000 bp promoter regions. Tissue-specific expression analysis found that high expressions of CsCDPK17 were in the mature leaves and seeds, while the lowest transcription in roots. The transcription of CsCDPK17 was increased during cold acclimation and decreased during de-acclimation procedures in four cultivars with different cold resistance abilities. Moreover, stress induced expression indicated that CsCDPK17 could be induced by cold, drought and osmotic stresses with the highest induction levels of 5.1, 2.3 and 2.4 folds, respectively. Overall, our results suggest that the newly cloned CsCDPK17 might be involve in the regulation of both development and abiotic stress responses (such as cold, drought and osmotic stress) in tea plants.

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