月季‘月月粉’RcDREB2A的克隆与表达分析

doi:10.16420/j.issn.0513-353x.2021-0794

摘要/Abstract

摘要：

以月季‘月月粉’（Rosa chinensis‘Old Blush’）扦插苗为试验材料,基于干旱胁迫的转录组克隆得到响应干旱胁迫的AP2/ERF类转录因子基因RcDREB2A,对其进行生物信息分析、表达分析以及转录因子特性分析。结果表明,RcDREB2A包含1 155 bp的开放阅读框,编码384个氨基酸,在75 ~ 142位氨基酸区域含有1个保守的AP2结构域;系统进化树分析表明RcDREB2A与野草莓（Fragaria vesca）FvDREB2A（XP_004307690.1）亲缘性最高;RcDREB2A蛋白预测分子量为5 737,理论等电点为4.75,无跨膜结构域,不含信号肽;定位于细胞核,没有转录激活活性;RcDREB2A在月季‘月月粉’叶片中表达量较高,且能被ABA、干旱、低温、盐胁迫处理诱导表达上调。以上结果表明,RcDREB2A可能参与月季多种非生物胁迫的响应。

关键词: 月季, RcDREB2A, 非生物胁迫, 转录因子

Abstract:

Cutting of Rosa chinensis‘Old Blush’were used as the material to clone the AP2/ERF transcription factor RCDREB2A gene based on transcriptome under drought stress. The RcDREB2A gene contained a 1 155 bp open reading frame（ORF）,encoding 384 amino acids and contained a conserved AP2 domain between 75-142 amino acids. Phylogenetic analysis showed that RcDREB2A has the highest affinity with Fragaria veaca FvDREB2A（XP_004307690.1）. RcDREB2A protein has a predicted molecular weight of 5 737,a theoretical isoelectric point of 4.75. RcDREB2A protein has no transmembrane domain and no signal peptide. RcDREB2A protein was localized in the nucleus and showed no transcriptional activation activity. RcDREB2A was highly expressed in leaves,and it could be up-regulated by ABA,drought,low temperature and salt stresses. The above results indicated that RcDREB2A may participate in a variety of abiotic stress responses of rose and plays a crucial role in resisting abiotic stresses.

Key words: rose, RcDREB2A, abiotic stress, transcription factor

中图分类号:

S685.12

贾鑫, 曾臻, 陈月, 冯慧, 吕英民, 赵世伟. 月季‘月月粉’RcDREB2A的克隆与表达分析[J]. 园艺学报, 2022, 49(9): 1945-1956.

JIA Xin, ZENG Zhen, CHEN Yue, FENG Hui, LÜ Yingmin, ZHAO Shiwei. Cloning and Expression Analysis of RcDREB2A Gene in Rosa chinensis‘Old Blush’[J]. Acta Horticulturae Sinica, 2022, 49(9): 1945-1956.

图/表 8

参考文献 35

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用途 Application	质粒名称 Plasmid name	引物序列（5′-3′） Primer sequence
扩增RcDREB2A全长 Amplification of RcDREB2A full length	RcDREB2A-F	ATGGGTGCTTATGATCAAGGTTCTAATGG
扩增RcDREB2A全长 Amplification of RcDREB2A full length	RcDREB2A-R	GTTTGCATTGCCGCCGCAGCCT
荧光定量PCR Quantitative Real-time PCR	RcDREB2A-F1	GAGCAGGCACATGACTGGATTAGC
荧光定量PCR Quantitative Real-time PCR	RcDREB2A-R1	TCTCGGAGCAGGGATGGGTTTAC
内参基因荧光定量PCR	RcPP2A-F	GAGGACAGGACCAGGAAGG
Internal reference genes Quantitative Real-time PCR	RcPP2A-R	GCTCTACGCCGCCAACAT
亚细胞定位 Subcellular localization	pBI121-RcDREB2A-GFP-F	CATTTACGAACGATACTCGAGATGGGTGCTTATGATCAAGGTTCTAATGG
亚细胞定位 Subcellular localization	pBI121-RcDREB2A-GFP-R	CACCATCACTAGTACGTCGACGTTTGCATTGCCGCCGCAGCCT
转录激活活性分析 Transcriptional activation activity analysis	pGBKT7-RcDREB2A-A-F	ATGGAGGCCGAATTCATGGGTGCTTATGATCAAGGTTC
	pGBKT7-RcDREB2A-A-R	CCGCTGCAGGTCGACGGATCCGTTTGCATTGCCGCCGCA
	pGBKT7-RcDREB2A-N-F	ATGGAGGCCGAATTCATGGGTGCTTATGATCAAGGTTC
	pGBKT7-RcDREB2A-N-R	CCGCTGCAGGTCGACGGATCCAAAGATTCCTCCTCTGCACAGACC
	pGBKT7-RcDREB2A-C-F	ATGGAGGCCGAATTCTATAGTGAAAACTGAGGATGGTGAGG
	pGBKT7-RcDREB2A-C-R	CAGGTCGACGGATCCGTTTGCATTGCCGCCGCA

用途 Application	质粒名称 Plasmid name	引物序列（5′-3′） Primer sequence
扩增RcDREB2A全长 Amplification of RcDREB2A full length	RcDREB2A-F	ATGGGTGCTTATGATCAAGGTTCTAATGG
扩增RcDREB2A全长 Amplification of RcDREB2A full length	RcDREB2A-R	GTTTGCATTGCCGCCGCAGCCT
荧光定量PCR Quantitative Real-time PCR	RcDREB2A-F1	GAGCAGGCACATGACTGGATTAGC
荧光定量PCR Quantitative Real-time PCR	RcDREB2A-R1	TCTCGGAGCAGGGATGGGTTTAC
内参基因荧光定量PCR	RcPP2A-F	GAGGACAGGACCAGGAAGG
Internal reference genes Quantitative Real-time PCR	RcPP2A-R	GCTCTACGCCGCCAACAT
亚细胞定位 Subcellular localization	pBI121-RcDREB2A-GFP-F	CATTTACGAACGATACTCGAGATGGGTGCTTATGATCAAGGTTCTAATGG
亚细胞定位 Subcellular localization	pBI121-RcDREB2A-GFP-R	CACCATCACTAGTACGTCGACGTTTGCATTGCCGCCGCAGCCT
转录激活活性分析 Transcriptional activation activity analysis	pGBKT7-RcDREB2A-A-F	ATGGAGGCCGAATTCATGGGTGCTTATGATCAAGGTTC
	pGBKT7-RcDREB2A-A-R	CCGCTGCAGGTCGACGGATCCGTTTGCATTGCCGCCGCA
	pGBKT7-RcDREB2A-N-F	ATGGAGGCCGAATTCATGGGTGCTTATGATCAAGGTTC
	pGBKT7-RcDREB2A-N-R	CCGCTGCAGGTCGACGGATCCAAAGATTCCTCCTCTGCACAGACC
	pGBKT7-RcDREB2A-C-F	ATGGAGGCCGAATTCTATAGTGAAAACTGAGGATGGTGAGG
	pGBKT7-RcDREB2A-C-R	CAGGTCGACGGATCCGTTTGCATTGCCGCCGCA

元件名称 Site name	序列 Sequence	元件数 Number of sites	功能 Function
ABRE	ACGTG	3	脱落酸响应元件Abscisic acid-responsive element
ATCT-motif	AATCTAATCC	1	光响应元件Light responsive element
CGTCA-motif	CGTCA	1	茉莉酸响应元件MeJA-responsive element
MYB	AAACCA	2	干旱诱导相关MYB结合位点MYB binding site involved in drought-inducibility
G-Box	CACGTT	2	光响应元件Light responsive element
GARE-motif	TCTGTTG	1	赤霉素响应元件Gibberellin-responsive element
LTR	CCGAAA	2	低温响应元件Low-temperature responsive element
P-box	CCTTTTG	1	赤霉素响应元件Gibberellin-responsive element
TATC-box	TATCCCA	1	赤霉素响应元件Gibberellin-responsive element
TGA-element	AACGAC	2	生长素响应元件Auxin-responsive element
TGACG-motif	TGACG	1	茉莉酸响应元件MeJA-responsive element

元件名称 Site name	序列 Sequence	元件数 Number of sites	功能 Function
ABRE	ACGTG	3	脱落酸响应元件Abscisic acid-responsive element
ATCT-motif	AATCTAATCC	1	光响应元件Light responsive element
CGTCA-motif	CGTCA	1	茉莉酸响应元件MeJA-responsive element
MYB	AAACCA	2	干旱诱导相关MYB结合位点MYB binding site involved in drought-inducibility
G-Box	CACGTT	2	光响应元件Light responsive element
GARE-motif	TCTGTTG	1	赤霉素响应元件Gibberellin-responsive element
LTR	CCGAAA	2	低温响应元件Low-temperature responsive element
P-box	CCTTTTG	1	赤霉素响应元件Gibberellin-responsive element
TATC-box	TATCCCA	1	赤霉素响应元件Gibberellin-responsive element
TGA-element	AACGAC	2	生长素响应元件Auxin-responsive element
TGACG-motif	TGACG	1	茉莉酸响应元件MeJA-responsive element