茶树生长素外运载体基因CsPIN3的克隆与表达分析

摘要

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 amplifi-cation of cDNA ends (RACE). The full length cDNA of CsPIN3 is 2654 bp (GenBank accession No. KP896474) and contained a 1926 bp open reading frame (ORF) encoding 641 amino acid residues. Bioinformatic analyses showed that CsPIN3 is not a secre-tory protein and had a molecular weight of 70.15 kD, atheoretical isoelectric point of 8.42. Subcellular localization prediction showed that CsPIN3 is 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 Arabi-dopsis 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 phase was higher than that at dormant phase (from initial dormant stage to expanding stage) and an obvious expression jump was detected at bud sprouting stage. These results demonstrated that CsPIN3 might be associated with the regulation of tea plant bud dormancy formation and break.%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、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PIN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。