Isolation and Function Analysis of the Promoter of a Thermal Inducible Gene RCA1 in Rhododendron hainanense

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (3): 566-576.doi: 10.16420/j.issn.0513-353x.2020-0483

• Research Papers • Previous Articles Next Articles

Isolation and Function Analysis of the Promoter of a Thermal Inducible Gene RCA1 in Rhododendron hainanense

LI Zheng, LIU Bing, ZHOU Hong, WANG Xiuyun(), XIA Yiping()

Genomics and Genetic Engineering Laboratory of Ornamental Plants,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China

Received:2020-09-04 Online:2021-03-25 Published:2021-04-02
Contact: WANG Xiuyun,XIA Yiping E-mail:wxy.550@163.com;ypxia@zju.edu.cn

Abstract

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

CLC Number:

S685.21

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.

Figures/Tables 9

References 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

Isolation and Function Analysis of the Promoter of a Thermal Inducible Gene RCA1 in Rhododendron hainanense

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