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作物学报 ›› 2016, Vol. 42 ›› Issue (01): 58-69.doi: 10.3724/SP.J.1006.2016.00058

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

茶树生长素外运载体基因CsPIN3的克隆与表达分析

王博1,2，曹红利1,2，黄玉婷1,2，胡玉荣1,2，钱文俊1,3，郝心愿1，王璐1，杨亚军1,*，王新超1,*   

  1. 1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；2中国农业科学院研究生院，北京 100081；3 西北农林科技大学, 陕西杨凌 712100
  • 收稿日期:2015-06-08 修回日期:2015-09-06 出版日期:2016-01-12 网络出版日期:2015-10-08
  • 通讯作者: 杨亚军, E-mail: yjyang@mail.tricaas.com, Tel: 0571-86650266; 王新超, E-mail: xcw75@mail.tricaas.com, Tel: 0571-86653162
  • 基金资助:

    本研究由国家自然科学基金项目(31370690)，国家现代农业产业技术体系建设专项(CARS-23)和中国农业科学院科技创新工程(CAAS-ASTIP-2014- TRICAAS)资助。

Cloning and Expression Analysis of Auxin Efflux Carrier Gene CsPIN3 in Tea Plant (Camellia sinensis)

WANG Bo1,2,CAO Hong-Li1,2,HUANG Yu-Ting1,2,HU Yu-Rong1,2,QIAN Wen-Jun1,3,HAO Xin-Yuan1, WANG Lu1,YANG Ya-Jun1,*,WANG Xin-Chao1,*   

  1. 1 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; 2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3 Northwest A&F University, Yangling 712100, China
  • Received:2015-06-08 Revised:2015-09-06 Published:2016-01-12 Published online:2015-10-08
  • Contact: 杨亚军, E-mail: yjyang@mail.tricaas.com, Tel: 0571-86650266; 王新超, E-mail: xcw75@mail.tricaas.com, Tel: 0571-86653162
  • Supported by:

    This research was supported by the National Natural Science Foundation of China (31370690), the Modern Agro-industry Technology Research System (CARS-23) and the Scientific Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2014-TRICAAS),

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摘要：

从实验室前期茶树冷驯化转录组测序结果中筛选拼接得到1条与其他植物PIN蛋白高度相似的EST序列，采用反转录PCR结合RACE技术从茶树中克隆到生长素外运载体基因PIN3的全长cDNA序列，命名为CsPIN3 (GenBank登录号为KP896474)。CsPIN3全长2654 bp，包含1926 bp的完整开放阅读框(ORF)，编码641个氨基酸；生物信息学分析显示，CsPIN3编码的蛋白质分子量为70.15 kD，理论等电点为8.42，是一种非分泌性蛋白；亚细胞定位显示，CsPIN3主要分布于质膜上，在内质网中有少量分布，是典型的膜蛋白；氨基酸序列分析表明CsPIN3编码蛋白由两端的疏水区和中间的亲水区构成。疏水区内有多个跨膜螺旋，其中N端疏水区有5个跨膜螺旋，C端有4个，与水稻的PIN蛋白结构相似。亲水区存在2个可变结构域，还存在着糖基化位点和磷酸化位点以及调控PIN蛋白内吞作用的NPNXY保守内在构型(Inner Motif, IM)，如PIN蛋白特有的丝氨酸/苏氨酸蛋白激酶(PID/PINOID)磷酸化活性位点——TPRXS(N/S)结构域；相似性及系统进化分析表明，该基因编码的氨基酸序列具有较高的保守性，与杨树、葡萄、柑橘、烟草、番茄、马铃薯、和芝麻等植物的PIN序列相似性在80%以上，与茄科植物的亲缘关系最近。在拟南芥PINs蛋白中，AtPIN3与茶树CsPIN3的亲缘关系较近。组织表达特异性分析表明 CsPIN3在茶树根、茎、叶、花中均有表达，在花中的表达量较高，在茎、叶中的表达量略高于根部。实时定量PCR分析显示，CsPIN3在龙井43茶树越冬芽萌发阶段的表达量高于休眠阶段(休眠初期到膨大期之间)，在茶芽萌动过程中表达上调的速度明显。推测该基因可能与茶树越冬芽休眠的维持和解除相关。

关键词: 茶树, 休眠, 生长素外运载体基因, 表达分析

Abstract:

On the basis of previous transcriptome study on tea plant cold acclimatization, we obtained a PIN homology gene named CsPIN3 and cloned its full-length cDNA sequence by reverse transcription-PCR (RT-PCR) combining with rapid amplification of cDNA ends (RACE). The full length cDNA of CsPIN3 was 2654 bp (GenBank accession No. KP896474) and contained a 1 926 bp open reading frame (ORF) encoding 641 amino acid residues. Bioinformatic analyses showed that CsPIN3 was not a secretory protein and had a molecular weight of 70.15 kD, atheoretical isoelectric point of 8.42. Subcellular localization prediction showed that CsPIN3 was a typical membrane protein mainly located in plasmalemma and then in endoplasmic reticulum. Moreover, Amino acid sequence analysis indicated that CsPIN3 protein contained hydrophobic regions in both ends and hydrophilic regions in the middle. Similar to PIN protein in rice, the hydrophobic regions of CsPIN3 consisted of several transmembrane helixes, among which five was in N motif and four in C motif. The hydrophilic regions of CsPIN3 had two unstable domains, several o-glycosylation sites, several phosphorylation sites like TPRXS (N/S) motif (a PID/PINOID phosphorylation site) and a well characterized conserved inner motif NPNXY regulating the endocytosis of PIN. Comparison of sequences similarity showed that the amino acid sequence coded by CsPIN3 had more than 80% similarity with reported PINs of Populus trichocarpa, Vitis vinifera, Citrus sinensis, Nicotiana tomentosiformis, Solanum lycopersicum, Solanum tuberosum, and Sesamum indicum. Phylogenetic tree analysis showed that CsPIN3 had the closest genetic relationship with Solanaceae and the highest identity with AtPIN3 of Arabidopsis thaliana PIN proteins. The CsPIN3 gene differentially expressed in different tea plant tissues, and transcript abundance in flower was much higher than that in leaf, stem and root. In addition, we analyzed the expression of CsPIN3 by qRT-PCR during the different phases of bud dormancy formation and break, and the results indicated that in cultivar Longjing 43, the expression level of CsPIN3 at growth stage was higher than that at dormant stage (initial dormant stage to expanding stage) and an obvious expression jump was detected at bud sprouting stage. These demonstrated that CsPIN3 could be associated with the regulation of tea plant bud dormancy formation and break.

Key words: Tea plant (Camellia sinensis), Dormancy, Auxin efflux carrier, Expression analysis

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