Cloning and Functional Analysis of PqDFR and PqANS Genes and Its Promoters from Paeonia qiui

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

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (6): 1256-1272.doi: 10.16420/j.issn.0513-353x.2023-0336

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Cloning and Functional Analysis of PqDFR and PqANS Genes and Its Promoters from Paeonia qiui

LI Qinqin, DONG Shanrong, LUO Jianrang^*(), ZHANG Yanlong

College of Landscape Architecture and Arts，Northwest A & F University，Yangling，Shaanxi 712100，China

Received:2024-02-23 Revised:2024-05-27 Online:2024-12-18 Published:2024-06-22
Contact: LUO Jianrang

Abstract

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

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.

Figures/Tables 16

References 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

Cloning and Functional Analysis of PqDFR and PqANS Genes and Its Promoters from Paeonia qiui

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