卵叶牡丹PqDFR和PqANS及启动子克隆与功能分析

doi:10.16420/j.issn.0513-353x.2023-0336

摘要/Abstract

摘要：

为探究卵叶牡丹（Paeonia qiui）春色叶呈现紫红色的成因，从其叶片转录组数据中筛选出两个花青素合成关键基因PqDFR和PqANS进行克隆、表达模式分析、功能鉴定以及启动子活性分析。PqDFR开放阅读框为1 095 bp，编码364个氨基酸，含有NADPH保守结构域和1个substract-binding结合位点；PqANS开放阅读框为1 065 bp，编码354个氨基酸，含有2OG-FeⅡ_Oxy和结合亚铁离子的保守氨基酸。实时荧光定量PCR结果表明，PqDFR和PqANS在叶片发育过程中表达量先上升再下降，与花青素的积累趋势基本一致。在卵叶牡丹叶盘中分别沉默PqDFR和PqANS基因后，叶盘的花青素含量均明显下降。稳定过表达试验表明转基因拟南芥幼苗相比野生型花青素积累更明显。PqDFR和PqANS启动子序列含有MYB转录因子结合序列，也含有光响应元件（G-box）、脱落酸响应元件（ABRE）、生长素响应元件（TGA-element）等顺式作用元件，GUS染色和定量结果显示启动子均具有良好的活性。研究结果表明PqDFR和PqANS可以促进卵叶牡丹叶片花青素合成。

关键词: 卵叶牡丹, 叶片, 花青素, 结构基因, 启动子

Abstract:

To explore the causes of the spring leaves of Paeonia qiui with purple-red color，PqDFR and PqANS genes were selected as key genes for anthocyanin synthesis based on the leaf transcriptional data. The two genes were cloned，analyzed for expression patterns，functional characterization and analyzed for promoter activity. The open reading frame of PqDFR was 1 095 bp，encoding 364 amino acids，containing the NADPH conserved domain and a substract-binding binding site. The open reading frame of PqANS was 1 065 bp，encoding 354 amino acids，including 2OG-FeⅡ_Oxy and conserved amino acids binding ferrous ion. The results of real-time quantitative PCR showed that the expression of PqDFR and PqANS genes showed a trend of increasing first，then decreasing during leaf development，which was consistent with the accumulation of anthocyanin. The anthocyanin content of leaf disks was decreased after silencing of PqDFR and PqANS genes in Paeonia qiui. Stable overexpression assays showed that transgenic Arabidopsis seedlings all showed significant anthocyanin accumulation compared to wild-type. Both PqDFR and PqANS gene promoters contain MYB binding sequence，light response elements（G-box），abscisic acid response element（ABRE）and growth factor response element（TGA-element）. GUS staining and quantitative results showed that both PqDFR and PqANS gene promoters had good activity. This research suggests PqDFR and PqANS can promote anthocyanin synthesis in Paeonia qiui leaves.

Key words: Paeonia qiui, leaf, anthocyanin, structural genes, promoter

李琴琴, 董山榕, 罗建让, 张延龙. 卵叶牡丹PqDFR和PqANS及启动子克隆与功能分析[J]. 园艺学报, 2024, 51(6): 1256-1272.

LI Qinqin, DONG Shanrong, LUO Jianrang, ZHANG Yanlong. Cloning and Functional Analysis of PqDFR and PqANS Genes and Its Promoters from Paeonia qiui[J]. Acta Horticulturae Sinica, 2024, 51(6): 1256-1272.

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参考文献 51

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引物名称 Name of primer	引物序列（5′-3′） Primer sequence
PqDFR	F：ATGGAAGCAGTGACCGAGTG；R：TTAGATTGTGTCATTAACAT
PqANS	F：ATGGTGAATTCAGTAGCTCC；R：TCAATTCTTAAACTCTTCTT
PqDFR（p1300）	F：GGGGTACCGCAAATGGAAGCAGTGACCGA；R：ACGCGTCGATGAAGTAGTGATCGACCCCTCT
PqANS（p1300）	F：ACGCGTCGACATGGTGAATTCAGTAGCTCCAAG；R：AACTGCAGCTGTATAAATCCATCACGGCCC
PqDFR（pTRV2）	F：TGCCAAAAGCGGATACCCAT；R：TCACTCCAGCAGGTTTCATC
PqANS（pTRV2）	F：CAACCAGCGAATACGCCAAG；R：GACACATTTTGCCGTCACCC
Actin	F：GGAACTGGAATGGTGAAGGCTG；R：CGATTGGATACTTCAGAGTGAGGA
PqDFR（qRT-PCR）	F：GCGGATACCCATTTGACCCT；R：TGAAAACGACTCTCCGCACA
PqANS（qRT-PCR）	F：TGAACTAGCTCTTGGCGTGG；R：CAGCACCGGCTTGAGTATGA

引物名称 Name of primer	引物序列（5′-3′） Primer sequence
PqDFR	F：ATGGAAGCAGTGACCGAGTG；R：TTAGATTGTGTCATTAACAT
PqANS	F：ATGGTGAATTCAGTAGCTCC；R：TCAATTCTTAAACTCTTCTT
PqDFR（p1300）	F：GGGGTACCGCAAATGGAAGCAGTGACCGA；R：ACGCGTCGATGAAGTAGTGATCGACCCCTCT
PqANS（p1300）	F：ACGCGTCGACATGGTGAATTCAGTAGCTCCAAG；R：AACTGCAGCTGTATAAATCCATCACGGCCC
PqDFR（pTRV2）	F：TGCCAAAAGCGGATACCCAT；R：TCACTCCAGCAGGTTTCATC
PqANS（pTRV2）	F：CAACCAGCGAATACGCCAAG；R：GACACATTTTGCCGTCACCC
Actin	F：GGAACTGGAATGGTGAAGGCTG；R：CGATTGGATACTTCAGAGTGAGGA
PqDFR（qRT-PCR）	F：GCGGATACCCATTTGACCCT；R：TGAAAACGACTCTCCGCACA
PqANS（qRT-PCR）	F：TGAACTAGCTCTTGGCGTGG；R：CAGCACCGGCTTGAGTATGA

引物名称 Name of primer	引物序列（5′-3′） Primer sequence
PqDFR-SP1	TCCACAAGGTCAAATGGGTATC
PqDFR-SP2	AAAAATAAAACCACCCCCACAG
PqDFR-SP3	GTCCCAGTAACAGGTGTAGATG
PqANS-SP1	AAACCTCCCCTGCTTTCTTAACC
PqANS-SP2	GTAAACGTTGCTGCAAAAATGG
PqANS-SP3	GCCCTTCTTCTTTCTTCTCTTCCTC
PBI121	F：TGGAAAGCGGGCAGTGAGCG；R：ATCCAGACTGAATGCCCACA
PqDFR（PBI121）	F：CCCAAGCTTAGGAGTAGCAAAGGCTGCACG；R：CGCGGATCCTTGTGTTTGGGTTCATGTTAGCTT
PqANS（PBI121）	F：CCCAAGCTTCATAGGCATTTATAATTAGTTAC；R：CGCGGATCCAGCAACGTTTACTCTCTGTTTTC

引物名称 Name of primer	引物序列（5′-3′） Primer sequence
PqDFR-SP1	TCCACAAGGTCAAATGGGTATC
PqDFR-SP2	AAAAATAAAACCACCCCCACAG
PqDFR-SP3	GTCCCAGTAACAGGTGTAGATG
PqANS-SP1	AAACCTCCCCTGCTTTCTTAACC
PqANS-SP2	GTAAACGTTGCTGCAAAAATGG
PqANS-SP3	GCCCTTCTTCTTTCTTCTCTTCCTC
PBI121	F：TGGAAAGCGGGCAGTGAGCG；R：ATCCAGACTGAATGCCCACA
PqDFR（PBI121）	F：CCCAAGCTTAGGAGTAGCAAAGGCTGCACG；R：CGCGGATCCTTGTGTTTGGGTTCATGTTAGCTT
PqANS（PBI121）	F：CCCAAGCTTCATAGGCATTTATAATTAGTTAC；R：CGCGGATCCAGCAACGTTTACTCTCTGTTTTC

调控序列 Regulatory sequence	序列 Sequence	数量 Number	特性 Characteristic
ARE	AAACCA	1	对厌氧诱导至关重要 Essential for the anaerobic induction
O2-site	GATGATGTGG	2	参与玉米醇溶蛋白代谢调控的顺式作用调控因子cis-Acting regulatory factors irnvolved in regulation of zein metabolism
TATA-box	TATA	4	转录起始位点 Transcription initiation site
CAAT-box	CAAAT	2	启动子和增强子调控元件 Common cis-acting element in promoter and enhancer regions
G-Box	CACGTG	2	参与光反应的元件 cis-Acting regulatory element involved in light responsiveness
GT1-motif	GGTTAA	1	参与光反应的元件 cis-Acting regulatory element involved in light responsiveness
ABRE	ACGTG	3	脱落酸反应顺式作用元件Abscisic acid reactive cis-acting element
TC-rich repeats	ATTCTCTAAC	1	参与防御和胁迫反应 Involved in defense and stress responsiveness
MYB	CAACCA	2	转录因子结合位点Transcription factor binding site
STRE	AGGGG	1	压力响应顺式调控元件Pressure responsive cis-control element
CCAAT-box	CAACGG	1	MYBHv1结合位点MYBHv1 binding site