Cloning of MYB Family Gene MawuCPC of Magnolia wufengensis and Analysis of Its Role in Flower Color Regulation

doi:10.7668/hbnxb.20194867

Abstract

Abstract:

Magnolia wufengensis is a rare ornamental tree species endemic to China.Its flower color variation types are extremely rich.There are a series of flower color variation varieties such as deep red,pink and white.However,the molecular mechanism of flower color regulation is still unclear.In order to explore the molecular mechanism of flower color regulation of Magnolia wufengensis,MawuCPC,a key candidate gene for flower color regulation of Magnolia wufengensis,was cloned from Magnolia wufengensis.Phylogenetic analysis,amino acid sequence structure analysis,subcellular localization experiment,yeast two-hybrid experiment and luciferase experiment were carried out.The expression level of MawuCPC in different flower color varieties was compared and analyzed by Real-time Quantitative PCR.The results showed that the coding region of MawuCPC gene was 258 bp,encoding 85 amino acids.Phylogenetic analysis showed that MawuCPC was clustered with CPC members of Arabidopsis thaliana and other species in the same evolutionary branch,which further supported that MawuCPC belonged to the CPC family of Magnolia wufengensis.Sequence structure analysis further showed that MawuCPC protein had only a conserved R3 domain and a bHLH binding motif.The result of subcellular localization experiment showed that MawuCPC protein was specifically localized in the nucleus.Yeast two-hybrid and luciferase assays showed that MawuCPC and MawuPAP1 could interact with the bHLH family protein MawubHLH1 of Magnolia wufengensis,and the binding of MawuCPC to MawubHLH1 could seriously inhibit the enhancement expression of MawuPAP1 and MawubHLH1 on the promoter of the structural gene MawuDFR gene.Real-time Quantitative PCR results showed that the expression level of MawuCPC was the highest in the white flower variety JB,followed by the pink flower variety Jiaoyan,and the lowest in the red flower variety Jiaohong 1.The above experimental results indicate that in Magnolia wufengensis,MawuCPC may also play its role in flower color regulation by competing with PAP1/2 protein to bind bHLH protein,and maybe the difference in the expression level of this gene promotes Magnolia wufengensis to produce extremely rich flower color variation types.

Key words: Magnolia wufengensis, MawuCPC, PCR, Yeast two-hybrid, Expression regulation, Flower color

WANG Qidi, ZHU Huixian, MA Jiang, CHEN Faju, ZHU Zhonglong, LI Xueping. Cloning of MYB Family Gene MawuCPC of Magnolia wufengensis and Analysis of Its Role in Flower Color Regulation[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 110-118. doi: 10.7668/hbnxb.20194867.

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References 26

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引物名称 Primer name	引物序列(5'—3') Primer sequences(5'—3')	引物用途 Primer use
MawuCPC-F	ACTTACATTGCTATGGATAAACGAC	基因克隆
MawuCPC-R	AAAGGCAGTTCCTTCAGCTATCACG
MawuCPC-qPCR-F	ATGGATAAACGACGCAGAAAGCA	荧光定量PCR
MawuCPC-qPCR-R	CCACTTGTCGCCTACGAGCCTA
MawuActin-F	AAGAACATCCCGTCCTCCTTACTG	内参基因
MawuActin-R	ACCGGAATCAAGCACAATACCTGT
MawuCPC-eGFP-F	TCGAGCTTTCGCAGTCTAGAATGGATAAACGACGCAGAAAGCAG	亚细胞定位载体构建
MawuCPC-eGFP-R	CCCTTGCTCAACCATTCTAGAGCTATCACGACCTTCTTTCTTCCC
MawuCPC-AD-F	GGAGGCCAGTGAATTCATGGATAAACGACGCAGAAAGCAGCC	酵母双杂交
MawuCPC-AD-R	CGAGCTCGATGGATCCTCAGCTATCACGACCTTCTTTCTTCCCG
MawuPAP1-AD-F	GGAGGCCAGTGAATTCATGGGTCAATCAGGAGTTCGAAAGG	酵母双杂交
MawuPAP2-AD-R	CGAGCTCGATGGATCCTCAGTGGCCATCTAATTGCGCCCAA
MawubHLH1-BD-F	CATGGAGGCCGAATTCATGGCATCCCCGCCGAGCAATCAGC	酵母双杂交
MawubHLH1-BD-R	GCAGGTCGACGGATCCCCAACCATATCAGTAATGGGGGATG
_proMawuDFR∷FLUC-F	ACCCTCCATGGTCTAGACCCAGCCAGATCGGATTAGGTACGATTC	荧光素酶试验
_proMawuDFR∷FLUC-R	TTTTTGGCGTCTTCCATCCCACCACTGGTCCCTTCATCTCTTC
_pro35S∷MawuCPC-F	GCTTTCGCAGTCTAGATGGATAAACGACGCAGAAAGCAGCC	荧光素酶试验
_pro35S∷MawuCPC-R	CGATCGTTAATTAAGTCAGCTATCACGACCTTCTTTCTTCCCG
_pro35S∷MawuPAP1-F	GCTTTCGCAGTCTAGATGGGTCAATCAGGAGTTCGAAAGG	荧光素酶试验
_pro35S∷MawuPAP1-R	CGATCGTTAATTAAGTCAGTGGCCATCTAATTGCGCCCAA
_pro35S∷MawubHLH1-F	GCTTTCGCAGTCTAGGAAGAGGGTGGTCTGGTGTAGATG	荧光素酶试验
_pro35S∷MawubHLH1-R	CGATCGTTAATTAAGCCAACCATATCAGTAATGGGGGATG

引物名称 Primer name	引物序列(5'—3') Primer sequences(5'—3')	引物用途 Primer use
MawuCPC-F	ACTTACATTGCTATGGATAAACGAC	基因克隆
MawuCPC-R	AAAGGCAGTTCCTTCAGCTATCACG
MawuCPC-qPCR-F	ATGGATAAACGACGCAGAAAGCA	荧光定量PCR
MawuCPC-qPCR-R	CCACTTGTCGCCTACGAGCCTA
MawuActin-F	AAGAACATCCCGTCCTCCTTACTG	内参基因
MawuActin-R	ACCGGAATCAAGCACAATACCTGT
MawuCPC-eGFP-F	TCGAGCTTTCGCAGTCTAGAATGGATAAACGACGCAGAAAGCAG	亚细胞定位载体构建
MawuCPC-eGFP-R	CCCTTGCTCAACCATTCTAGAGCTATCACGACCTTCTTTCTTCCC
MawuCPC-AD-F	GGAGGCCAGTGAATTCATGGATAAACGACGCAGAAAGCAGCC	酵母双杂交
MawuCPC-AD-R	CGAGCTCGATGGATCCTCAGCTATCACGACCTTCTTTCTTCCCG
MawuPAP1-AD-F	GGAGGCCAGTGAATTCATGGGTCAATCAGGAGTTCGAAAGG	酵母双杂交
MawuPAP2-AD-R	CGAGCTCGATGGATCCTCAGTGGCCATCTAATTGCGCCCAA
MawubHLH1-BD-F	CATGGAGGCCGAATTCATGGCATCCCCGCCGAGCAATCAGC	酵母双杂交
MawubHLH1-BD-R	GCAGGTCGACGGATCCCCAACCATATCAGTAATGGGGGATG
_proMawuDFR∷FLUC-F	ACCCTCCATGGTCTAGACCCAGCCAGATCGGATTAGGTACGATTC	荧光素酶试验
_proMawuDFR∷FLUC-R	TTTTTGGCGTCTTCCATCCCACCACTGGTCCCTTCATCTCTTC
_pro35S∷MawuCPC-F	GCTTTCGCAGTCTAGATGGATAAACGACGCAGAAAGCAGCC	荧光素酶试验
_pro35S∷MawuCPC-R	CGATCGTTAATTAAGTCAGCTATCACGACCTTCTTTCTTCCCG
_pro35S∷MawuPAP1-F	GCTTTCGCAGTCTAGATGGGTCAATCAGGAGTTCGAAAGG	荧光素酶试验
_pro35S∷MawuPAP1-R	CGATCGTTAATTAAGTCAGTGGCCATCTAATTGCGCCCAA
_pro35S∷MawubHLH1-F	GCTTTCGCAGTCTAGGAAGAGGGTGGTCTGGTGTAGATG	荧光素酶试验
_pro35S∷MawubHLH1-R	CGATCGTTAATTAAGCCAACCATATCAGTAATGGGGGATG

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