CN110923213A - 一种茶树蛋白激酶基因CsCIPK序列及其应用 - Google Patents

一种茶树蛋白激酶基因CsCIPK序列及其应用 Download PDF

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CN110923213A
CN110923213A CN201811117987.XA CN201811117987A CN110923213A CN 110923213 A CN110923213 A CN 110923213A CN 201811117987 A CN201811117987 A CN 201811117987A CN 110923213 A CN110923213 A CN 110923213A
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cscipk
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tea tree
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庄静
沈威
刘昊
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Abstract

本发明利用聚合酶扩增技术从茶树‘龙井43’中克隆得到一个蛋白激酶基因CsCIPK，其序列包含1341个核苷酸，编码446个氨基酸。序列分析表明，CsCIPK蛋白含有丝氨酸/苏氨酸激酶结构域和NAF结构域。表达分析结果表明，CsCIPK蛋白参与了多种逆境胁迫的响应，在高温、干旱及盐处理4h、低温处理24h时达到最高。本发明从茶树‘龙井43’中克隆获得CsCIPK基因，该基因可调控植物逆境胁迫响应过程。

Description

一种茶树蛋白激酶基因CsCIPK序列及其应用
技术领域
本发明属于基因工程领域，涉及一个茶树蛋白激酶基因及其应用。本发明公开了一种利用聚合酶扩增技术从茶树‘龙井43’中克隆出蛋白激酶基因CsCIPK的方法，该基因可用于茶树逆境胁迫响应机制研究。
背景技术
茶树是我国一种重要的经济作物，栽培历史悠久，作为一种叶用植物，茶叶中富含茶多酚、氨基酸、维生素、有机酸、矿物质等次生代谢成分，是一种集营养和保健为一体的天然饮料(宛晓春，茶叶生物化学，中国农业出版社，2003)。茶树在生长发育中会受到多种非生物胁迫的伤害，其中高温、低温、干旱、高盐等严重影响茶叶的产量和品质。因此，提高茶树的抗逆性，对于茶树的推广种植，具有十分重要的意义。
CIPK基因能够参与多种植物抗逆途径，且在不同非生物逆境下表达呈现差异。在拟南芥中，AtCIPK7与CBL1结合成复合体，在抵御低温胁迫中发挥作用(黄聪琳，博士论文，兰州大学，2011)。AtCIPK6在植物盐胁迫和生长素运输中发挥着重要作用(Tripathi V等，Plant Journal for Cell&Molecular Biology，2009，58(5)：778-790)。在水稻中，OsCIPK1受低温诱导表达强烈，并且在幼叶中表达量最高(Kim等，Plant Molecular Biology，2003，52(6)：1191-1202)。过量表达OsCIPK3基因，能够显著提高转基因水稻对低温的抗性(Xiang等，Plant Physiology，2007，144(3)：1416-1428)。
发明内容
本发明提供了一种茶树蛋白激酶基因CsCIPK基因的制备方法和用途。所获得的茶树CsCIPK基因能够应用于作物抗逆品种的研究与培育。
附图说明
图1.茶树CsCIPK蛋白的保守域预测结果
图2.茶树与其他植物CIPK蛋白的系统进化树分析
图3.茶树CsCIPK基因在不同非生物胁迫下的表达分析
具体实施方式
1.植物材料：本发明所用植物材料为茶树品种‘龙井43’，植株种植于南京农业大学茶叶科学研究所。
2.总RNA提取及cDNA合成：按照植物总RNA提取试剂盒RNA Quick RNA IsolationKit(北京华越洋公司)提取茶树‘龙井43’叶片的总RNA。测定样品RNA浓度后利用反转录试剂盒Prime Script with gDNA Eraser(大连TaKaRa生物科技有限公司)合成cDNA。
3.茶树CsCIPK基因的克隆：基于本实验室茶树转录组测序结果(Wu等，BMC PlantBiology，2014，14：277)和茶树基因组数据(Xia等，Molecular Plant，2017，10(6)：866-877)，检索到一个茶树CIPK基因序列，并设计一对扩增引物。正向引物序列为5’-ATGGTGGTGAGAAAGGTTG-3’，反向引物序列为5’-TCAATGCTTTTTGTTCTTATT-3’。以茶树‘龙井43’cDNA为模板，利用聚合酶链式反应扩增目的片段，扩增程序为：95℃5min；94℃30s，55℃30s，72℃90s，共30个循环；最后72℃延伸10min。经琼脂糖凝胶电泳检测并回收扩增产物后，将其连接至pMD19-T载体再转化到大肠杆菌DH5a中，菌液检测后由南京金斯瑞生物科技有限公司完成测序。
4.序列分析：相关核苷酸和氨基酸序列下载及CsCIPK蛋白保守域预测在NCBI网站(http：//ncbi.nlm.nih.gov)中完成；氨基酸多序列比对由ClustalW(https：//www.genome.jp/tools-bin/clustalw)完成。
5.茶树CsCIPK基因转录水平分析：根据克隆所得茶树CsCIPK基因序列，设计荧光定量引物，正向引物序列为5’-TGGACGATGTGAATGCTGTT-3’，反向引物序列为5’-GAATCCTTCCGACGATCAAA-3’。选用茶树GAPDH基因作为内参基因，按照SYBR Premix Ex Taq试剂盒(大连TaKaRa公司)操作说明来进行实时荧光定量PCR(quantitative real timeRT-PCR)。采用2-ΔΔCT方法进行相对定量计算，数据分析在IBM SPSS Statistic和WPS Excel软件中完成。
Figure ISA0000171155360000011

Claims (5)

1.一种从茶树中获得的蛋白激酶基因CsCIPK。
2.权利要求1所述的蛋白激酶基因CsCIPK的核苷酸序列。
3.一种权利要求1所述源于茶树的CsCIPK基因的制备方法，具体包括以下步骤：
1)基于本实验室茶树转录组测序结果，检索茶树中蛋白激酶基因CsCIPK核苷酸序列；
2)设计克隆引物，正向：5’-ATGGTGGTGAGAAAGGTTG-3’，反向：5’-TCAATGCTTTTTGTTCTTATT-3’，通过聚合酶扩增技术从茶树品种‘龙井43’中克隆获得CsCIPK基因。
4.权利要求1所述的茶树中蛋白激酶基因CsCIPK的功能研究：通过实时荧光定量PCR技术，测定茶树基因CsCIPK在不同逆境胁迫下的表达水平。
5.权利要求1所述的茶树中蛋白激酶基因CsCIPK的应用。
CN201811117987.XA 2018-09-19 2018-09-19 一种茶树蛋白激酶基因CsCIPK序列及其应用 Pending CN110923213A (zh)

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