茶树生长素外运载体基因CsPIN3的克隆与表达分析

发布时间：2019-02-18 08:56

【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(Gen Bank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,At PIN3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。
[Abstract]:A highly similar EST sequence to other plant PIN proteins was obtained from the sequencing of cold acclimated transcriptome of tea plants in early laboratory. The full-length cDNA sequence of auxin gene PIN3 was cloned from tea plant by reverse transcription PCR and RACE technique, named CsPIN3 (Gen Bank accession number (KP896474). The CsPIN3 full-length 2654 bp, contains a complete open reading frame (ORF), (ORF), containing 1926 bp. Encoding 641 amino acids. Bioinformatics analysis showed that the molecular weight of the protein encoded by CsPIN3 was 70.15 kD, and the theoretical isoelectric point was 8.42, which was a non-secretory protein. Subcellular localization showed that CsPIN3 was mainly distributed on the plasma membrane and a small amount in the endoplasmic reticulum (ER), which was a typical membrane protein. Amino acid sequence analysis showed that the CsPIN3 encoded protein was composed of hydrophobic region at both ends and hydrophilic region in the middle. There are many transmembrane helices in the hydrophobic region, among which there are 5 transmembrane helices in N-terminal hydrophobic region and 4 in C-terminal, which are similar to the PIN protein structure of rice. There were two variable domains in the hydrophilic region, glycosylation sites and phosphorylation sites, and NPNXY conserved intrinsic configuration (inner motif,IM), which regulated the endocytosis of PIN protein. For example, TPRXS (N / S) domain, the phosphorylated site of serine / threonine protein kinase (PID/PINOID) specific to PIN protein; Similarity and phylogenetic analysis showed that the amino acid sequence of the gene was highly conserved, and the PIN sequence similarity with poplar, grape, citrus, tobacco, tomato, potato and sesame was more than 80%. There is a close relationship with the plants of the family Solanaceae. In Arabidopsis thaliana PIN protein, the relationship between At PIN3 and CsPIN3 is close. Tissue expression specificity analysis showed that CsPIN3 was expressed in roots, stems, leaves and flowers of tea plants, but higher in flowers, and slightly higher in stems and leaves than in roots. Real-time quantitative PCR analysis showed that the expression of CsPIN3 in the germination stage of overwintering bud of Longjing 43 tea plant was higher than that in dormancy stage (from the early dormancy stage to the expanding stage), and the rate of up-regulation of CsPIN3 expression was obvious in the process of tea bud germination. It is speculated that this gene may be related to the maintenance and release of dormancy in overwintering buds of tea plants.
【作者单位】：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;中国农业科学院研究生院;西北农林科技大学;
【基金】：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) 中国农业科学院科技创新工程(CAAS-ASTIP-2014-TRICAAS)资助~~
【分类号】：S571.1

【参考文献】