Cloning and Functional Characterization of Chalcone Synthase Genes（CmCHS）from Clivia miniata

doi:10.16420/j.issn.0513-353x.2021-0090

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (10): 1847-1858.doi: 10.16420/j.issn.0513-353x.2021-0090

• Research Papers • Previous Articles Next Articles

Cloning and Functional Characterization of Chalcone Synthase Genes（CmCHS）from Clivia miniata

LIU Yue¹, LI Yueqing², MENG Xiangyu³, HUANG Jing¹, TANG Hao¹, LI Yuanheng¹, GAO Xiang², ZHAO Chunli¹^,^*()

¹College of Horticulture,Jilin Agricultural University,Changchun 130118,China
²Key Laboratory of Molecular Epigenetics of Ministry of Education,Northeast Normal University,Changchun 130024,China
³College of Life Science and Bioengineering,Shenyang University,Shenyang 110044,China

Received:2021-06-09 Revised:2021-08-30 Online:2021-10-25 Published:2021-11-01
Contact: ZHAO Chunli E-mail:zcl8368@163.com

Abstract

Abstract:

In order to investigate the structure and function of Chalcone Synthase from Clivia miniata,members of CHS gene family were screened and identified based on the previously constructed transcriptome database. The results showed that four CmCHSs（CmCHS1,CmCHS2,CmCHS3 and CmCHS4）genes were cloned in C. miniata. The open reading frames of the four CmCHSs were 1 173,1 170,1 173 and 1 173 bp and encoding 390,389,390,390 respectively. Amino acid sequence that CmCHSs had typical the conserved domains. Phylogenetic tree analysis showed that the four CmCHS genes clustered with the bona-fide CHSs from other plants and belonged to type Ⅲ polyketide synthase（PKS）super family. The results of subcellular localization showed that they were localized in the cytoplasm and nucleus. Gene expressionanalysis showed CmCHS1 and CmCHS2 were highly expressed at the late stage of flower development process,and the transcripts of CmCHS1,CmCHS2 and CmCHS3 were confirmed to the extensively detected in pigmented leaves and red fruit peels. In contrast,the expression level of CmCHS4 gene was relatively low. In order to firmly verified the roles of CmCHSs in the biosynthesis of anthocyanins,prokaryotic expression vectors,pET-32-CmCHS,were constructed and recombinant proteins were prepared and purified,which were subjected to in vitro enzyme activity analysis. Results showed that the four CmCHS recombinant proteins could catalyze coumarinyl-CoA and malonyl-Coenzyme A into naringenin chalcone. This study explored the bioinformatics analysis,gene expression analysis,and functional analysis of CmCHS genes,and laid a theoretical foundation for the genetic transformation and flower color improvement of C. miniata.

Key words: Clivia miniata, flavonoid, anthocyanin, chalcone synthase, enzyme activity

CLC Number:

S682.13

LIU Yue, LI Yueqing, MENG Xiangyu, HUANG Jing, TANG Hao, LI Yuanheng, GAO Xiang, ZHAO Chunli. Cloning and Functional Characterization of Chalcone Synthase Genes（CmCHS）from Clivia miniata[J]. Acta Horticulturae Sinica, 2021, 48(10): 1847-1858.

Figures/Tables 6

References 49

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用途 Use	基因 Gene	引物序列（5′-3′） Primer sequence
基因ORF的克隆 Cloning the full-length of ORF	CmCHS1	F:ATGGTGAGTATCGATGAGATT;R:CATCGCGCCGATGATTAATT
	CmCHS2	F:ATGGTGAGTGTCCATGAGATTCGC;R:CACGATGTGTCGCATCGATAAATT
	CmCHS3	F:ATGGTGAGCGTGGAAGAGATC;R:GGTATCACACCGATGATTAATT
	CmCHS4	F:ATGGCCACAATTGAGGAGATCAG;R:ACGCTTCACACCGCTAGTTAACT
	T7	F:TAATACGACTCACTATAGGG;R:GCTAGTTATTGCTCAGCGG
实时荧光定PCR Quantitative Real-time PCR	CmCHS1	F:GACATGCCCGGTGCAGACTACCA;R:AGCCGTGATCTCCGAGCAGACG
	CmCHS2	F:GACGAGACAAGTGTTGAGCGAGTA;R:TCCAGGTCCGAATCCAAAGAGC
	CmCHS3	F:AGGAGCGATAGACGGGCACTTA;R:GAACAGCGAGTTCCAATCTAACATC
	CmCHS4	F:GGAGGTGCTGAAGGAGTATGGGAACA;R:TCCAGGGCCGAACCCAAATAGA
	CmActin	F:GCATCACACCTTCTACAA;R:CATTGTAGAAGGTGTGATG
制备重组蛋白 Preparation of recombinant protein	CmCHS1	F:TGGCTGATATCGGATCCATGGTGAGTATCGATGAG; R:CGACGGAGCTCGAATTCTTAATTAGTAGCCGCGCT
	CmCHS2	F:TGGCTGATATCGGATCCATGGTGAGTGTCCATGAG; R:CGACGGAGCTCGAATTCTTAAATAGCTACGCTGTG
	CmCHS3	F:TGGCTGATATCGGATCCATGGTGAGCGTGGAAGAG; R:CGACGGAGCTCGAATTCTTAATTAGTAGCCACACT
	CmCHS4	F:TGGCTGATATCGGATCCATGGCCACAATTGAGGAG; R:CGACGGAGCTCGAATTCTCAATTGATCGCCACACT
亚细胞定位 Subcellular	GFP(35S:GFP)	F:CTGATTACGCTCATATGATGGTGAGCAAGGGCGAG; R:AGGATTCAATCTTAAGTTACTTGTACAGCTCGTCCATG
localization	(GFP)CmCHS1	F:GACGAGCTGTACAAGCATATGGTGAGTATCGAT; R:CAACAGGATTCAATCTTAAGTTAATTAGTAGCCGC
	(GFP)CmCHS2	F:GACGAGCTGTACAAGCATATGGTGAGTGTCCAT; R:CAACAGGATTCAATCTTAAGTTAAATAGCTACGCT
	(GFP)CmCHS3	F:GACGAGCTGTACAAGCATATGGTGAGCGTGGAA; R:CAACAGGATTCAATCTTAAGTTAATTAGTAGCCAC
	(GFP)CmCHS4	F:GACGAGCTGTACAAGCATATGGCCACAATTGAG; R:CAACAGGATTCAATCTTAAGTCAATTGATCGCCAC

用途 Use	基因 Gene	引物序列（5′-3′） Primer sequence
基因ORF的克隆 Cloning the full-length of ORF	CmCHS1	F:ATGGTGAGTATCGATGAGATT;R:CATCGCGCCGATGATTAATT
	CmCHS2	F:ATGGTGAGTGTCCATGAGATTCGC;R:CACGATGTGTCGCATCGATAAATT
	CmCHS3	F:ATGGTGAGCGTGGAAGAGATC;R:GGTATCACACCGATGATTAATT
	CmCHS4	F:ATGGCCACAATTGAGGAGATCAG;R:ACGCTTCACACCGCTAGTTAACT
	T7	F:TAATACGACTCACTATAGGG;R:GCTAGTTATTGCTCAGCGG
实时荧光定PCR Quantitative Real-time PCR	CmCHS1	F:GACATGCCCGGTGCAGACTACCA;R:AGCCGTGATCTCCGAGCAGACG
	CmCHS2	F:GACGAGACAAGTGTTGAGCGAGTA;R:TCCAGGTCCGAATCCAAAGAGC
	CmCHS3	F:AGGAGCGATAGACGGGCACTTA;R:GAACAGCGAGTTCCAATCTAACATC
	CmCHS4	F:GGAGGTGCTGAAGGAGTATGGGAACA;R:TCCAGGGCCGAACCCAAATAGA
	CmActin	F:GCATCACACCTTCTACAA;R:CATTGTAGAAGGTGTGATG
制备重组蛋白 Preparation of recombinant protein	CmCHS1	F:TGGCTGATATCGGATCCATGGTGAGTATCGATGAG; R:CGACGGAGCTCGAATTCTTAATTAGTAGCCGCGCT
	CmCHS2	F:TGGCTGATATCGGATCCATGGTGAGTGTCCATGAG; R:CGACGGAGCTCGAATTCTTAAATAGCTACGCTGTG
	CmCHS3	F:TGGCTGATATCGGATCCATGGTGAGCGTGGAAGAG; R:CGACGGAGCTCGAATTCTTAATTAGTAGCCACACT
	CmCHS4	F:TGGCTGATATCGGATCCATGGCCACAATTGAGGAG; R:CGACGGAGCTCGAATTCTCAATTGATCGCCACACT
亚细胞定位 Subcellular	GFP(35S:GFP)	F:CTGATTACGCTCATATGATGGTGAGCAAGGGCGAG; R:AGGATTCAATCTTAAGTTACTTGTACAGCTCGTCCATG
localization	(GFP)CmCHS1	F:GACGAGCTGTACAAGCATATGGTGAGTATCGAT; R:CAACAGGATTCAATCTTAAGTTAATTAGTAGCCGC
	(GFP)CmCHS2	F:GACGAGCTGTACAAGCATATGGTGAGTGTCCAT; R:CAACAGGATTCAATCTTAAGTTAAATAGCTACGCT
	(GFP)CmCHS3	F:GACGAGCTGTACAAGCATATGGTGAGCGTGGAA; R:CAACAGGATTCAATCTTAAGTTAATTAGTAGCCAC
	(GFP)CmCHS4	F:GACGAGCTGTACAAGCATATGGCCACAATTGAG; R:CAACAGGATTCAATCTTAAGTCAATTGATCGCCAC

Cloning and Functional Characterization of Chalcone Synthase Genes（CmCHS）from Clivia miniata

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