牡丹PsGAI基因的生物信息学及原核表达分析

迟田钰,盖树鹏,刘春英,袁延超,孙阳,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:张玉喜;E-mail: zhang-yuxi@163.com

摘 要:

充足的低温累积和外源赤霉素(GA)处理是反季节催花的常用技术手段, 探究内休眠解除的机理是解决催花不良的前提。前期研究表明, GA途径的激活是牡丹(Paeonia suffruticosa)花芽内休眠解除的关键环节, 而DELLA蛋白是GA信号转导中的负调控因子。本研究从牡丹花芽转录组中筛选出DELLA家族成员中类GAI的EST序列, 进行了生物信息学、表达模式和原核表达分析。同源性比对结果表明牡丹类GAI与拟南芥AtGAI序列同源性最高, 全长cDNA为2 445 bp, 编码区1 848 bp, 共编码616个氨基酸, 蛋白分子质量为67.26 kDa。实时定量PCR分析表明其在低温诱导的牡丹花芽休眠解除中呈极显著下调。为分析其在蛋白水平上的变化趋势, 将PsGAI构建到含MBP的pMAL-c5X原核表达载体上, 转化至大肠杆菌BL21 (DE3)进行诱导表达。在18°C、IPTG浓度为1 mmol·L−1 的条件下诱导12 h, SDS-PAGE电泳检测MBP-PsGAI融合蛋白分子质量为112 kDa左右。实验结果为进一步纯化PsGAI蛋白及研究其在牡丹休眠解除中的作用提供理论依据。


关键词:牡丹; 内休眠; DELLA; 原核表达

收稿:2020-06-30   修定:2020-09-21

资助:国家自然科学基金(31872145和31972452)和国家重点研发计划(2018YFD1000403)

Bioinformatics and prokaryotic expression analysis of PsGAI in tree peony

CHI Tianyu, GAI Shupeng, LIU Chunying, YUAN Yanchao, SUN Yang, ZHANG Yuxi*
Key Lab of Plant Biotechnology in Universities of Shandong Province, College of Life Science, Qingdao Agricultural University, Qingdao, Shandong 266109, China

Corresponding author: ZHANG Yuxi; E-mail: zhang-yuxi@163.com

Abstract:

Sufficient low temperature accumulation and exogenous gibberellins (GAs) application are common techniques used to off-season flower forcing, and exploring the mechanism of endo-dormancy break is the premise to solve productive problems. Previous studies had shown that the activation of GA pathway was the key to break endo-dormancy in tree peony (Paeonia suffruticosa), and DELLA protein is the negative regulator of GA signal transduction. In this study, an EST sequence of GAI-like was obtained from the transcriptome, and bioinformatics analysis, expression pattern and prokaryotic expression were carried out. Phylogenetic analysis showed that the putative GAI-like protein had the highest homology with AtGAI. The full-length cDNA of PsGAI was 2 445 bp, containing a coding region of 1 848 bp encoding 616 amino acids with a calculated molecular mass of 26 kDa. Real-time quantitative PCR analysis showed that it was significantly downregulated during the chilling-induced bud dormancy release. In order to analyze its variation trend at the protein level, PsGAI was constructed on the prokaryotic expression vector of MBP and transformed into BL21 (DE3). After 12 h, the 112 kDa fusion protein expression was detected by SDS-PAGE electrophoresis at 18°C and under 1 mmol·L−1 IPTG. These results would provide theoretical basis for further purification of PsGAI protein and study its role during the dormancy release in tree peony.


Key words: Paeonia suffruticosa; endo-dormancy; DELLA; prokaryotic expression

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