海南杜鹃热诱导基因RhRCA1启动子的克隆与功能分析

doi:10.16420/j.issn.0513-353x.2020-0483

摘要/Abstract

摘要：

为了阐述RhRCA1基因的调控机制,以海南杜鹃（Rhododendron hainanense）3年生扦插苗为材料,分析RhRCA1高温响应和组织表达特性;并克隆获得RhRCA1的启动子序列,将该启动子分别驱动荧光素酶报告基因在烟草中瞬时表达、GUS报告基因在拟南芥中稳定表达,分析该启动子活性、组织特异性和热诱导性。结果显示:（1）25 ℃条件下,RhRCA1表达量极低,37 ℃处理后其表达量显著升高,呈现典型的热诱导特性,并且RhRCA1在绿色组织中的表达量显著高于非绿色组织,呈现组织特异性;（2）获得的启动子长1 624 bp,其含有多个非生物胁迫响应、光响应、组织特异性等相关元件;（3）构建RhRCA1启动子和萤光素酶融合的植物表达载体,在烟草叶片中瞬时表达,荧光成像结果表明RhRCA1启动子能强烈响应高温胁迫;（4）构建RhRCA1启动子和GUS融合的植物表达载体,转化拟南芥植株筛选至T₃代,GUS染色结果显示,高温能显著诱导RhRCA1启动子在拟南芥子叶、成熟叶、茎、萼片、果荚等绿色组织中的表达。研究结果表明RhRCA1启动子是1个兼具高温诱导型和组织特异性的启动子,可应用于植物抗逆基因工程,提高植物在高温胁迫下的抗性。

关键词: 杜鹃, Rubisco活化酶, 启动子, 热诱导, 功能分析

Abstract:

In order to analysis the regulatory mechanism and expression profile of RhRCA1,three-year-old cutting seedlings of Rhododendron hainanense were used as the experimental material to isolate the promoter ofRhRCA1. This promoter was then used to drive the Luciferase and GUS reporters in tobacco and Arabidopsis thaliana respectively,to figure out its activity,tissue specificity and thermal inducibility. The results showed that:(1) Remarkable high expression of RhRCA1 was observed in the quantitative experiment after high temperature treatment,which appeared typical thermal inducibility. (2) The promoter of RhRCA1 with a length of 1 624 bp was cloned and the promoter contained multiple cis-elements related to abiotic stress response,light signals and tissue specificity. (3) A plant expression vector was constructed using theRhRCA1 promoter to drive Luciferase report gene,and was transformed into tabacco to analyse transient expression. The results showed that RhRCA1promoter could strongly respond to heat stress. (4) The promoter and GUSfusion expression vector were constructed and transformed intoArabidopsis. Analysis of T₃ generation transgenic Arabidopsis tissues with histochemical staining showed that heat stress had a significant impact on the induction of RhRCA1 promoter expression in green tissues,including cotyledon,mature leaf,young stem,sepal and seedpods. These results indicated that the promoter ofRhRCA1is a heat-inducible and tissue-specific promoter. This promoter is ideal to drive other genes to improve the thermostability of plants under high temperature by genetic engineering,which has promising applications under the tendency of global warming.

Key words: Rhododendron hainanense, Rubisco activase, promoter, thermal inducibility, function analysis

中图分类号:

S685.21

李铮, 刘冰, 周泓, 王秀云, 夏宜平. 海南杜鹃热诱导基因RhRCA1启动子的克隆与功能分析[J]. 园艺学报, 2021, 48(3): 566-576.

LI Zheng, LIU Bing, ZHOU Hong, WANG Xiuyun, XIA Yiping. Isolation and Function Analysis of the Promoter of a Thermal Inducible Gene RCA1 in Rhododendron hainanense[J]. Acta Horticulturae Sinica, 2021, 48(3): 566-576.

图/表 9

参考文献 35

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名称 Name			序列 Sequence（5′-3′）
prRCA1-F prRCA1-R			ACTCGGCACAGCTACTACC CAAAGGTGGAAACGGCAG
RCA1-F RCA1-R 18S-F 18S-R			TGCTGGTTCAAGAGCAGGAG GTTGAGCTGCTTTGCCATAGA CGCATTCCCCACTGTATTAGAC CGTAACAAGGTTTCCGTAGGTG
prRCA1-F-Hind Ⅲ prRCA1-R-Nco I prRCA1-LUC-F prRCA1-LUC-R			ACCTGCAGGCATGCAAGCTTCTACTACCAAGCACCTCCGC TTACCCTCAGATCTACCATGGAGAAATCAAGGGTCTGTTTGGGA GCTTGATATCGAATTCCTGCAGCTACTACCAAGCACCTCCGC GGATCCCCCGGGCTGCAGAGAAATCAAGGGTCTGTTTGGGA
P3301-F-Hind Ⅲ P3301-R-Nco Ⅰ pGreen-LUC-F pGreen-LUC-R			ACCTGCAGGCATGCAAGCTT TTACCCTCAGATCTACCATGG GGCGATTAAGTTGGGTAACGC GGTTCCATCTTCCAGCGGATA
P3301-F-Hind Ⅲ GUS-R			ACCTGCAGGCATGCAAGCTT CACGGGTTGGGGTTTCTACA

名称 Name			序列 Sequence（5′-3′）
prRCA1-F prRCA1-R			ACTCGGCACAGCTACTACC CAAAGGTGGAAACGGCAG
RCA1-F RCA1-R 18S-F 18S-R			TGCTGGTTCAAGAGCAGGAG GTTGAGCTGCTTTGCCATAGA CGCATTCCCCACTGTATTAGAC CGTAACAAGGTTTCCGTAGGTG
prRCA1-F-Hind Ⅲ prRCA1-R-Nco I prRCA1-LUC-F prRCA1-LUC-R			ACCTGCAGGCATGCAAGCTTCTACTACCAAGCACCTCCGC TTACCCTCAGATCTACCATGGAGAAATCAAGGGTCTGTTTGGGA GCTTGATATCGAATTCCTGCAGCTACTACCAAGCACCTCCGC GGATCCCCCGGGCTGCAGAGAAATCAAGGGTCTGTTTGGGA
P3301-F-Hind Ⅲ P3301-R-Nco Ⅰ pGreen-LUC-F pGreen-LUC-R			ACCTGCAGGCATGCAAGCTT TTACCCTCAGATCTACCATGG GGCGATTAAGTTGGGTAACGC GGTTCCATCTTCCAGCGGATA
P3301-F-Hind Ⅲ GUS-R			ACCTGCAGGCATGCAAGCTT CACGGGTTGGGGTTTCTACA

类型 Type	调控元件 Regulated element	基序 Motif sequence	数量 Amount	生物学功能 Biological function
非生物胁迫响应元件 Abiotic stress responsive element	ARE	AAACCA	1	厌氧诱导必需的调控元件 cis-acting regulatory element essential for the anaerobic induction
	TC-rich repeats	GTTTTCTTAC	1	防御和胁迫响应元件cis-acting element involved in defense and stress responsiveness
	ABRE	ACGTG	5	脱落酸响应元件cis-acting element involved in the abscisic acid responsiveness
	RGAANNTTC	GGGAGTTTC	1	HSF1的结合位点 HSF1 binding site
	CCAAT-box	CCAAT	2	热激蛋白基因表达元件cis-element for heat shock protein gene expression
组织特异性相关元件 Tissue-specific element	CACTFTPPCA1	CACT	20	叶肉特异性表达元件cis-regulatory element for mesophyll-specific gene expression
	TGACGTVMAMY	TGACGT	2	子叶特异性表达元件cis-element for Expression in cotyledons of germinated seeds
	RBCSCONSENSUS	AATCAA	1	光调节和叶特异性表达元件cis-element for light-regulated and leaf-specific expression
光响应元件 Light responsive element	Box 4	ATTAAT	4	参与部分光响应保守DNA组件 Part of a conserved DNA module involved in light responsiveness
	G-box	CACGTC/ACACGTGGC/ TGACACGTGGCTCT	5	参与光反应的元件 cis-acting regulatory element involved in light responsiveness
	AE-box	AGAAACTT	1	部分光响应组件 Part of a module for light response
基础元件 Basal element	CAAT-box	CAAT/TGCCAAC	28	启动子和增强子调控元件 Common cis-acting element in promoter and enhancer regions
	TATA-box	TATA/TACAAAA	11	转录起始位点 Transcription initiation site
其他 Others	TGACG-motif	TGACG	5	参与茉莉酸甲酯诱导表达cis-acting regulatory element involved in the MeJA-responsiveness

类型 Type	调控元件 Regulated element	基序 Motif sequence	数量 Amount	生物学功能 Biological function
非生物胁迫响应元件 Abiotic stress responsive element	ARE	AAACCA	1	厌氧诱导必需的调控元件 cis-acting regulatory element essential for the anaerobic induction
	TC-rich repeats	GTTTTCTTAC	1	防御和胁迫响应元件cis-acting element involved in defense and stress responsiveness
	ABRE	ACGTG	5	脱落酸响应元件cis-acting element involved in the abscisic acid responsiveness
	RGAANNTTC	GGGAGTTTC	1	HSF1的结合位点 HSF1 binding site
	CCAAT-box	CCAAT	2	热激蛋白基因表达元件cis-element for heat shock protein gene expression
组织特异性相关元件 Tissue-specific element	CACTFTPPCA1	CACT	20	叶肉特异性表达元件cis-regulatory element for mesophyll-specific gene expression
	TGACGTVMAMY	TGACGT	2	子叶特异性表达元件cis-element for Expression in cotyledons of germinated seeds
	RBCSCONSENSUS	AATCAA	1	光调节和叶特异性表达元件cis-element for light-regulated and leaf-specific expression
光响应元件 Light responsive element	Box 4	ATTAAT	4	参与部分光响应保守DNA组件 Part of a conserved DNA module involved in light responsiveness
	G-box	CACGTC/ACACGTGGC/ TGACACGTGGCTCT	5	参与光反应的元件 cis-acting regulatory element involved in light responsiveness
	AE-box	AGAAACTT	1	部分光响应组件 Part of a module for light response
基础元件 Basal element	CAAT-box	CAAT/TGCCAAC	28	启动子和增强子调控元件 Common cis-acting element in promoter and enhancer regions
	TATA-box	TATA/TACAAAA	11	转录起始位点 Transcription initiation site
其他 Others	TGACG-motif	TGACG	5	参与茉莉酸甲酯诱导表达cis-acting regulatory element involved in the MeJA-responsiveness