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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2023, Vol. 49 Issue (1): 23-30    DOI: 10.3785/j.issn.1008-9209.2022.01.112
生物科学与技术     
拟南芥热激转录因子HSFB2b负调控植物热形态建成
姚紫薇1(),孙婧靓2,刘建祥1(),芦海平1()
1.浙江大学生命科学学院，浙江 杭州 310058
2.大连民族大学环境与资源学院，辽宁 大连 116600
Heat shock transcription factor HSFB2b negatively regulates plant thermomorphogenesis in Arabidopsis
Ziwei YAO1(),Jingliang SUN2,Jianxiang LIU1(),Haiping LU1()
1.College of Life Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China
2.College of Environment and Bioresources, Dalian Minzu University, Dalian 116600, Liaoning, China
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摘要：

为探究参与极端高温环境应答的热激转录因子（heat shock transcription factor, HSF）是否也参与了温和高温下的植物热形态建成，本研究通过CRISPR/Cas9基因编辑技术，结合一系列生理生化和遗传学实验，以及效应因子-报告基因系统（effector-reporter）等实验方法，发现热激转录因子HSFB2b受温和高温诱导，在热形态建成过程中发挥着重要作用。在温和高温（29 ℃）下，拟南芥突变体hsfb2b表现出比野生型更长的下胚轴表型，揭示HSFB2b作为负调控因子在热形态建成中发挥作用。亚细胞定位实验表明，HSFB2b蛋白在细胞核中发挥功能。实时荧光定量聚合酶链反应分析表明，温和高温下野生型植株中热激蛋白（heat shock proteins, HSPs）基因、热激转录因子HSFA2和茉莉酸降解基因ST2A相对于常温（22 ℃）时表达上调，但这些基因在突变体hsfb2b中上调倍数更大。此外，效应因子-报告基因系统实验证实HSFB2b可以结合ST2A启动子热激元件（heat shock element, HSE），从而抑制ST2A表达。综上所述，受温和高温诱导的热激转录因子HSFB2b在下胚轴伸长调控中起负调控作用，并且在分子机制上HSFB2b通过识别HSE元件，负调控茉莉酸降解相关基因ST2A的表达，从而负调控植物热形态建成。
关键词： 拟南芥;  热形态建成;  热激转录因子;  茉莉酸代谢;  下胚轴伸长    
Abstract:

In order to explore whether heat shock transcription factor (HSF) known to be involved in plant adaptation to extreme heat stress is also involved in plant thermomorphogenesis at warm temperatures, the result of CRISPR/Cas9 gene editing, physiological and biochemical, genetic experiments, and effector-reporter assay demonstrated that the heat shock transcription factor HSFB2b was induced by the warm temperature and played an important role in the process of plant thermomorphogenesis. Under the warm temperature (29 ℃), the Arabidopsis mutant hsfb2b exhibited a longer hypocotyl than the wild type, suggesting that HSFB2b functioned as a negative regulator in thermomorphogenesis. Subcellular localization results showed that the HSFB2b protein was localized in the nucleus. Real-time quantitative polymerase chain reaction (qRT-PCR) analysis showed that the heat shock proteins (HSPs) gene, the heat shock transcription factor HSFA2, and the jasmonic acid degradation gene ST2A were up-regulated in the wild type under the warm temperature relative to the normal temperature (22 ℃), but these genes were more up-regulated by the warm temperature in the hsfb2b mutant than that in the wild type. Furthermore, effector-reporter assay demonstrated that HSFB2b could inhibit ST2A expression by binding to the heat shock element (HSE). In conclusion, the heat shock transcription factor HSFB2b induced by the warm temperature played a negative regulatory role in the hypocotyl elongation and negatively regulated the expression of gene ST2A by recognizing the HSE in molecular mechanism, thusnegatively regulated the plant thermomorphogenesis.
Key words: Arabidopsis    thermomorphogenesis    heat shock transcription factor    jasmonic acid catabolism    hypocotyl elongation
收稿日期: 2022-01-11 出版日期: 2023-03-07
CLC:  Q943.2  
基金资助: 中央高校基本科研业务费专项资金(2021XZZX023);国家自然科学基金项目(31872653)
通讯作者: 刘建祥,芦海平     E-mail: 21907011@zju.edu.cn;jianxiangliu@zju.edu.cn;luhaiping@zju.edu.cn
作者简介: 姚紫薇（https://orcid.org/0000-0001-5259-585X），E-mail：21907011@zju.edu.cn
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引用本文:

姚紫薇,孙婧靓,刘建祥,芦海平. 拟南芥热激转录因子HSFB2b负调控植物热形态建成[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(1): 23-30.

Ziwei YAO,Jingliang SUN,Jianxiang LIU,Haiping LU. Heat shock transcription factor HSFB2b negatively regulates plant thermomorphogenesis in Arabidopsis. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(1): 23-30.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.01.112    或    https://www.zjujournals.com/agr/CN/Y2023/V49/I1/23

基因

Gene

编号

Code

引物序列（5′→3′）

Primer sequence (5′→3′)

HSFB2aAT5G62020F: CTATCCCAACGCCGTTTCTC
R: CGAAATCTGTCGGATTCCATACGA
HSFB2bAT4G11660F: TTGTTCGTCAGCTCAATACTTACG
R: TTTCCGCCGTTGAATATCCCGAA
HSFA2AT2G26150F: GCGGCTTCTTCATCTGTA
R: CTGTTACGACCATTACTCCAA
ST2AAT5G07010F: ATCCCAAGTTTCTCCATGTG
R: AACATCTCTTGGAACTCGCA
HSP17.6AAT1G59860F: ATGGGATCCATTCAAGGAACTT
R: TTCTTTCTTCATCCCCGGTAAA
HSP17.8AT1G07400F: CTCAAGATCAGTGGAGAGAGAC
R: CTTCTTCTTAGCCTCTTCCACT
HSP23.6AT4G25200F: ATGGCATCTGCTCTCGCTCT
R: AGGAACAGAGCGGCGATATA
PP2AAT1G13320F: TAACGTGGCCAAAATGATGC
R: GTTCTCCACAACCGCTTGGT
表1  qRT-PCR实验中所用引物
图1  温和高温下同源基因 HSFB2a 和 HSFB2b 的表达情况A. 拟南芥中HSFB亚家族蛋白质序列的同源性比对；B～C. HSFB2a及HSFB2b在不同温度下的表达量。n=3。
图2  HSFB2b 在拟南芥热形态建成中的负调控作用A～B. hsfb2a和hsfb2b的突变序列；C～D. 拟南芥下胚轴表型和长度。WT：野生型。短栅上不同小写字母表示在P＜0.05水平差异有统计学意义，下同。
图3  HSFB2a及HSFB2b蛋白的亚细胞定位
图4  温和高温下 HSFB2b 下游基因表达情况
图5  HSFB2b 负调控热形态建成A. 效应因子-报告基因载体构建示意图；B. 相对荧光素酶活性；C. HSFB2b调控植物热形态建成的工作模型图。*表示在P＜0.05水平差异有统计学意义。
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