《植物生理学报》 2021, 57(3): 558-568

水稻OsHDAC15基因的克隆、 表达分析及原核表达

杜巧丽¹，方远鹏¹，蒋君梅¹，李向阳²，谢鑫^1,*

¹贵州大学农学院农业微生物特色重点实验室, 贵阳550025；²贵州大学绿色农药与农业生物工程教育部重点实验室, 贵阳550025

通信作者：谢鑫；E-mail: xiexin2097757@163.com

摘　要：

组蛋白去乙酰化酶(histone deacetylase, HDAC)在植物生长发育、器官构建、逆境胁迫及激素信号应答中发挥重要作用, 并作为调节染色质结构和基因表达的关键表观遗传因素。其中HDAC15蛋白是HDAC家族中第II亚族(HDA) a类成员, 在植物应对非生物胁迫、病原物侵染等方面具有重要作用。本研究克隆了一个水稻(Oryza sativa) HDAC15基因, 该基因全长1 611 bp, 编码536个氨基酸, 含50个磷酸化激酶作用位点和一个Hist deacetyl结构域, 亚细胞定位于细胞核。系统发育分析表明, 该基因编码蛋白与短柄草(Brachypodium distachyon)蛋白同源性最高。互作预测分析表明, HDAC15基因可被多种miRNA作用, 从而实现对HDAC15基因功能表达的调控。实时荧光定量PCR(quantitative real-time PCR, RT-qPCR)分析表明, OsHDAC15基因的表达具有组织特异性; 在40°C高温胁迫下, OsHDAC15的表达水平在较短时间内受到诱导表达; 在干旱(300 mmol·L^–1, D-mannitol)、盐胁迫(250 mmol·L–1, NaCl)和外源脱落酸(200 µmol·L^–1, abscisic acid, ABA)处理条件下, 基因表达量均显著上调。 OsHDAC15基因同时也受激素吲哚乙酸(10 µmol·L^–1, indole-3-acetic acid, IAA)和4°C低温的诱导表达, 峰值出现相对滞后, 但最高表达量为对照组的27倍。同时为获得HDAC15可溶性蛋白, 将其转化到大肠杆菌(Escherichia coli) Rosetta (DE3)中进行原核表达, 结果显示, HDAC15蛋白最佳诱导温度和IPTG (异丙基-β-D-硫代吡喃半乳糖苷)浓度分别为30°C和0.8 mmol·L^–1。该研究为HDAC15基因参与水稻组蛋白修饰, 及其在组蛋白去乙酰化中的作用研究提供了证据。

关键词：水稻; OsHDAC15; 基因克隆; 表达分析

收稿：2020-10-22   修定：2020-12-03

资助：国家自然科学基金(31801691和32060614)、贵州省科技计划(黔科合支撑[2019]2408号)和贵州省高层次留学人才创新创业择优资 助项目[(2018)02号]

Cloning, expression analysis and prokaryotic expression of rice OsHDAC15 gene

DU Qiaoli¹, FANG Yuanpeng¹, JIANG Junmei¹, LI Xiangyang², XIE Xin^1,*

¹Key Laboratory of Agricultural Microbiology, College of Agriculture, GuiZhou University, Guiyang 550025, China; ²Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China

Corresponding author: XIE Xin; E-mail: xiexin2097757@163.com

Abstract:

Histone deacetylase (HDAC) plays an important role in plant growth and development, organ construction, adversity stress and hormone signal response, and is used as a key epigenetic factor regulating chromatin structure and gene expression. Among them, the HDAC15 protein is a member of the second subfamily (HDA) class a of the HDAC family, and plays an important role in the response of plants to abiotic stress and pathogen infection. In this study, a rice HDAC15 gene was cloned. The gene is 1 611 bp in length, encodes 536 amino acids, contains 50 phosphorylated kinase sites and a Hist deacetyl domain. The subcellular location is in the nucleus. The phylogenetic tree shows that it has the highest homology with Brachypodium distachyon. Interaction prediction analysis shows that HDAC15 gene can be acted on by a variety of miRNAs, so as to realize the regulation of HDAC15 gene functional expression. Quantitative real-time PCR (RT-qPCR) analysis shows that the expression of OsHDAC15 gene is tissue-specifc; under 40℃ high temperature stress, the expression level of OsHDAC15 is induced in a short time; in drought simulation (300 mmol·L^-1, D-mannitol), salt stress (250 mmol·L^-1, NaCl) and exogenous abscisic acid (200 µmol·L^-1, abscisic acid, ABA) treatment conditions, gene expression are signifcantly increased. OsHDAC15 gene is also induced by the hormone indole-3-acetic acid (10 µmol·L^-1, IAA) and 4℃ low temperature. The peak value is relatively lagging, but the highest expression level is 27 times that of the control group. At the same time, in order to obtain HDAC15 soluble protein, it was transformed into Escherichia coli Rosetta (DE3) for prokaryotic expression analysis. The results showed that the optimal induction temperature and IPTG concentration of HDAC15 protein were 30℃ and 0.8 mmol·L^-1, respectively. This study provides evidence for the involvement of HDAC15 gene in rice histone modifcation and deacetylation.

Key words: rice; OsHDAC15; gene cloning; expression analysis