《植物生理学报》 2016, 52(8): 1280-1286

牡丹丙酮酸脱氢酶基因PsPDH的克隆和表达特性分析

杨丽金^*, 甘甜^*, 盖树鹏, 刘春英, 张玉喜^**

青岛农业大学生命科学学院/山东省高校植物生物技术重点实验室, 山东青岛266109

通信作者：杨丽金；E-mail: zhang-yuxi@163.com

摘　要：

根据454测序得到的丙酮酸脱氢酶基因PDH的部分cDNA片段设计引物, 运用cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)技术扩增得到牡丹(Paeonia suffruticosa) PDH基因的全长cDNA, 命名为PsPDH。PsPDH cDNA序列全长为1 313 bp, 包括132 bp的5′非编码区、173 bp的3′非编码区和1 008 bp的编码区, 共编码335个氨基酸, 其分子量为35.844 kDa。推测的氨基酸序列包括17个可能的磷酸化位点, 其中包括12个Ser位点, 这可能与活性位点的调节有关。亚细胞定位预测该蛋白位于线粒体基质中。同源性比对表明, PsPDH与葡萄(Vitis vinifera) VvPDH同源性最高, 与拟南芥(Arabidopsis thaliana) AtPDH的相似性最低。实时定量PCR (quantitative real-time PCR)结果表明PsPDH基因在初花期叶片、根和心皮中表达水平较高, 在人工低温处理14 d的牡丹花芽中表达水平最高, PDH的酶活力变化趋势与该基因表达趋势一致, 可见, PsPDH基因的活化可能是促进花芽休眠解除的原因。本研究为深入解析休眠解除中呼吸代谢的调控提供参考。

关键词：PsPDH; 克隆; 生物信息学分析; 表达模式; 酶活力

收稿：2016-06-07   修定：2016-07-25

资助：国家自然科学基金(31471908和31372104)。 * 并列第一作者。

Molecular cloning and expression pattern analysis of pyruvate dehydrogenase PsPDH gene from tree peony (Paeonia suffruticosa)

YANG Li-Jin^*, GAN Tian^*, GAI Shu-Peng, LIU Chun-Ying, ZHANG Yu-Xi^**

College of Life Sciences, Qingdao Agricultural University / Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao, Shandong 266109, China

Corresponding author: YANG Li-Jin; E-mail: zhang-yuxi@163.com

Abstract:

According to the partial fragment of pyruvate dehydrogenase gene PDH obtained from 454 sequencing, the full-length cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR, and named as PsPDH. The full-length cDNA of PsPDH was 1 313 bp including 132 bp 5′ untranslated region (UTR), 173 bp 3′UTR and 1 008 bp encoding frame, which encodes 335 amino acids with the molecular weight of 35.844 kDa. The result of phosphorylation site predication shows that the PsPDH protein had 17 possible phosphorylation sites including 12 Ser sites, which might be associated with the regulation of active sites. The result of subcellular prediction indicates that PsPDH was mainly located in mitochondrial matrix. Alignment analysis was consistent with phylogenetic analysis, which indicates that PsPDH was highly conserved with VvPDH, lowest with AtPDH, and basically accorded with botanical classification system. The results of quantitative real-time PCR indicate that the PsPDH transcripts were abundant in the leaf, root and carpel at the early stage of flowering, and were highest in flower buds after 14 d chilling duration. The relative activity of PDH was consistent with the expression pattern of PsPDH gene during dormancy release. Taken together, the activation of PsPDH transcripts might be a critical cue to promote dormancy release of tree peony, and the results provide theoretical basis to validate the regulation mechanism of respiratory metabolism during dormancy release.

Key words: PsPDH; cloning; bioinformatics analysis; expression patterns; enzyme activity