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作物学报 ›› 2016, Vol. 42 ›› Issue (03): 376-388.doi: 10.3724/SP.J.1006.2016.00376

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

茶树中性/碱性转化酶基因CsINV10的克隆与表达分析

钱文俊1,2,岳川2,曹红利2,郝心愿2,王璐2,王玉春1,2,黄玉婷2,王博2,王新超2,肖斌1,*,杨亚军1,2,*   

  1. 1西北农林科技大学园艺学院,陕西杨凌 712100;2中国农业科学院茶叶研究所 / 国家茶树改良中心 / 农业部茶树生物学与资源利用重点实验室,浙江杭州 310008
  • 收稿日期:2015-08-12 修回日期:2015-11-20 出版日期:2016-03-12 网络出版日期:2015-12-18
  • 通讯作者: 杨亚军, E-mail: yjyang@mail.tricaas.com, Tel: 0571-86653162; 肖斌, E-mail: xiaobin2093@sohu.com
  • 基金资助:

    本研究由国家自然科学基金项目(31170650), 国家现代农业产业技术体系建设专项(CARS-23), 浙江省自然科学基金项目(LY14C160001),浙江省农业新品种选育重大专项(2012C2905-3)和中国农业科学院科技创新工程项目(CAAS-ASTIP-2014-TRICAAS)资助。

Cloning and Expression Analysis of a Neutral/alkaline Invertase Gene (CsINV10) in Tea Plant (Camellia sinensis L. O. Kuntze)

QIAN Wen-Jun1,2,YUE Chuan2,CAO Hong-Li2,HAO Xin-Yuan2,WANG Lu2,WANG Yu-Chun1,2,HUANG Yu-Ting2,WANG Bo2,WANG Xin-Chao2,XIAO Bin1,*,YANG Ya-Jun1,2,*   

  1. 1 College of Horticulture, Northwest Agriculture and Forestry University, Yangling 712100, China; 2 Tea Research Institute of Chinese Academy of Agricultural Sciences / National Center for Tea Improvement / Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Hangzhou 310008, China
  • Received:2015-08-12 Revised:2015-11-20 Published:2016-03-12 Published online:2015-12-18
  • Contact: 杨亚军, E-mail: yjyang@mail.tricaas.com, Tel: 0571-86653162; 肖斌, E-mail: xiaobin2093@sohu.com
  • Supported by:

    This study was supported by the National Natural Science Foundation of China (31170650), the Earmarked Fund for China Agriculture Research System (CARS-23), the Natural Science Foundation of Zhejiang Province (LY14C160001), the Major Project for New Agricultural Varieties Breeding of Zhejiang Province (2012C2905-3) and the Chinese Academy of Agricultural Sciences through an Innovation Project for Agricultural Sciences and Technology (CAAS-ASTIP-2014-TRICAAS).

摘要:

基于课题组前期对茶树冷驯化系统的转录组测序分析结果,从中挑选出6条与中性/碱性转化酶基因高度相似的EST序列,电子拼接和RT-PCR验证后获得一条全长为2101 bp的核酸序列。该基因包含1923 bp的ORF,编码640个氨基酸,蛋白分子量为71. 8 kD,理论等电点(pI)为5.69。根据BlastX同源性比对显示该基因与荔枝LcNI相似性最高(80%),为G100家族成员,属于中性/碱性转化酶基因,将其命名为CsINV10(GenBank登录号为KT359348)。对CsINV10氨基酸序列的系统进化树分析显示,其与木薯MeNINV8亲缘关系最近。进一步分析显示,CsINV10的氨基酸序列无N端信号肽,无跨膜结构域,属于亲水性蛋白,并定位在叶绿体上。荧光定量PCR分析表明,CsINV10具有组织表达特异性,在茶树叶和花中的表达量最高,根系中最低。分析发现,低温(4℃)、干旱和盐胁迫分别处理茶树1 d后,成熟叶片中CsINV10的表达呈逐渐上升趋势;而在ABA条件处理下,该基因呈先升高后降低趋势,在处理5 d后基本不表达,表明该基因可能参与茶树对多种逆境胁迫的响应,这为后续进一步研究转化酶基因在茶树抗寒等逆境胁迫中的作用奠定基础。

关键词: 茶树中性, 碱性转化酶基因, 定量分析

Abstract:

Based on the comprehensive RNA-Seq analysis of tea plant during cold acclimation stage, we picked out six ESTs sequences with a high similarity to neutral/alkaline invertase gene to get a splicing. As a result, a full-length of 2101 bp nucleotide sequence was obtained from tea plant after validated by using RT-PCR technique. Bioinformatics analysis showed that the sequence containing 1923 bp ORF (Open Reading Fram) and encoding 640 amino acid residues with a putative molecular mass of 71.8 kD and theoretical isoelectric point of 5.69, was named as CsINV10 (GenBank accession number: KT359348). BlastX and phylogenetic analysis indicated that the protein encoded by CsINV10 shared the highest identity (80%) with LcNI in Litchi,and had a closest genetic relationship with MeNINV8 in Manihot esculenta Crantz. Also, CsINV10 as a neutral/alkaline invertase gene could be classified into G100 family. The protein had no N-terminal signal peptide and transmembrane domain, and was predicated to be a hydrophilic protein localized in chloroplast. The expression analysis of CsINV10 under different abiotic stress conditions showed that cold, PEG and salt stresses could gradually promote the expression of CsINV10 when treated for one day, however, it had a transient increase when treated by ABA after three hours. Consequently, we speculated that CsINV10 might be involved in the tea plant response to abiotic stresses. Moreover, we found that CsINV10 had a tissue-specific expression patterns in leaf and flower. This study will provide a theoretical basis for the functional analysis of invertase genes of tea plant in response to various stresses.

Key words: Tea plant (Camellia sinensis), Neutral/alkaline invertase gene, Expression analysis

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