Identification and Function Analysis of AcMYB88 in Kiwifruit （Actinidia chinensis）

doi:10.13560/j.cnki.biotech.bull.1985.2023-0726

Abstract

Abstract:

【Objective】 MYB transcription factors play an important role in anthocyanin accumulation of kiwifruit. Identifying MYB transcription factors that regulate anthocyanin biosynthesis and verifying gene functions facilitate elucidating the molecular mechanism of anthocyanin accumulation.【Method】 It was reported that AcMYB110 was a key transcription factor promoting anthocyanin accumulation in kiwifruit. In PlantTFDB database, 12 MYB genes that showing high homology to AcMYB110 were identified from 97 kiwifruit MYB genes by phylogenetic tree analysis. Bioinformatics methods were employed to analyze their physical and chemical properties, hydrophilicity and hydrophobicity, protein phosphorylation sites, protein secondary structures, and phylogenetic relationships with other species.【Result】 The 12 MYB transcription factors encoded amino acids ranging from 200 to 423; their molecular weight ranged from 22.3 to 45.5 kD, and all of them were hydrophilic proteins located in the nucleus. The majority of potential phosphorylation sites in the 12 candidate MYB proteins were found to be at serine residues. Moreover, their secondary structures were primarily composed of random coils with some α-helices. Among the 12 MYB transcription factors, the expression pattern of AcMYB88 gene was very similar to that of AcMYB110 gene during kiwifruit development.【Conclusion】 Transient overexpression experiments in tobacco demonstrate that AcMYB88 acts as a positive regulator of anthocyanin accumulation, and AcMYB88 synergistically promotes anthocyanin accumulation with AcMYB110 and AcbHLH42. This study provides a theoretical foundation for understanding anthocyanin biosynthesis and breeding research in kiwifruit

Key words: Actinidia chinensis, MYB, family analysis, anthocyanins

YUAN Xin-yu, ZHONG Cai-hong, ZHANG Long, ZHENG Hao, LI Ji-tao, ZHANG Qiong. Identification and Function Analysis of AcMYB88 in Kiwifruit （Actinidia chinensis）[J]. Biotechnology Bulletin, 2024, 40(2): 183-196.

Figures/Tables 14

References 35

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基因名称 Gene name	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）	备注 Note
Actin	TGAGAGATTCCGTTGCCCAGAAGT	TTCCTTACTCATGCGGTCTGCGAT	实时荧光定量PCR分析所用引物 Primers for real-time RT-qPCR analysis
Achn033551	GCATCCATGACATCACCAGC	AGCATAACCGGTGTCCCAAC
Achn036201	TGACCGGTGGTACAATGTGG	CCTGATGGTTCACCCACTCC
Achn141271	GGGCTCTCGCGATATACGAC	TCTCCAACGTGAACGACGAG
Achn145641	TCCATGACATCACCAGCGTC	AGGAAGCATAACTGGCGTCC
Achn154591	ACGACATCACCACCGTCAAT	GCCGGTCCTTCACTTGGTAG
Achn172831	GGAGTGCTCCTCGGATTGTC	CCACTTTCTGCCACCGATCA
Achn207851	CTGTGGTACCCAGAATGCCT	ATGTCGTGGATGCTTGACCG
Achn212251	AAGGCCAACCGTGAATCTCC	AGGCAATCCGACATGGGTTC
Achn218481	AGAAGTACTTCATCCGGCAGC	GGAATTGCGTTCTTGGATTGG
Achn258201	CCCTGGCAACTTATCCCGAG	CTTGCGTCGCTCCTGATCTA
Achn365361	ACGACGGCAGTGATTATCGG	CTTGTCCTCGAACCTCGTCC
Achn371351	ATTGGAGGAGCATATCGCGG	TGGATGCTTGAACGTCTCCC
Acc00493（AcMYB10）	GCCATCACCACCATCCAACA	TTCCCCAAAATTGGCTCCTCA
Acc19563（AcbHLH42）	TCGTTTAGGGCCGAGTCTAC	TTGAGAGTAGTGCGTGTCGT
pSAK-Achn365361	ACTAGTGGATCCAAAGAATTCATGTATGGCGACGACGGC	AGAAGTACTCTCGAGAAGCTTTACATGGGATAGTTGAAGTTTTGGTACCC	过表达载体构建所用引物 Primers used for overexpression vector construction
pSAK-AcMYB10	ACTAGTGGATCCAAAGAATTCATGGAAAGTGTTACTTTAGGAGTGAGAAAGG	AGAAGTACTCTCGAGAAGCTTTAATCATCACCTAAGAGATCCCAAAGGTTAACA
pSAK-AcbHLH42	ACTAGTGGATCCAAAGAATTCATGGCGGCTCCCCCT	AGAAGTACTCTCGAGAAGCTTCAGGGTATGATTTGGTGTATTGCTCTCT
NtActin	AATGGAACTGGAATGGTCAAGGC	TGCCAGATCTTCTCCATGTCATCCCA	烟草RT-qPCR分析所用引物 Primers for real-time RT-qPCR analysis in tobacco
AcMYB88	ACGACGGCAGTGATTATCGG	CTTGTCCTCGAACCTCGTCC
AcMYB10	GCCATCACCACCATCCAACA	TTCCCCAAAATTGGCTCCTCA
AcbHLH42	TCGTTTAGGGCCGAGTCTAC	TTGAGAGTAGTGCGTGTCGT
NtC4H	ACAACCCGGATCACTGGAAG	ACTGTCCACCTTTCTCCGTG
NtCHS	TTGTTCGAGCTTGTCTCTGC	AGCCCAGGAACATCTTTGAG
NtPAL	TTACGCCCTTCGAACATCCC	GAGGCAAGTGCAAGCCTAGT
Nt4CL	ACTGCCGAAAGGTGTGATGT	AAATCGCTGCTCCGACTCTC
NtCHI	GTCAGGCCATTGAAAAGCTC	CTAATCGTCAATGCCCCAAC
NtF3H	CAAGGCATGTGTGGATATGG	TGTGTCGTTTCAGTCCAAGG
NtDFR	AACCAACAGTCAGGGGAATG	TTGGACATCGACAGTTCCAG
NtANS	TATCCCAAATGCCCCCAACC	ATCTTCTCCTTTGGCGGCTC
NtANR	TTCCATCTCTCATGGCTGGTC	ATATGGGCGCGACAAACATC

基因名称 Gene name	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）	备注 Note
Actin	TGAGAGATTCCGTTGCCCAGAAGT	TTCCTTACTCATGCGGTCTGCGAT	实时荧光定量PCR分析所用引物 Primers for real-time RT-qPCR analysis
Achn033551	GCATCCATGACATCACCAGC	AGCATAACCGGTGTCCCAAC
Achn036201	TGACCGGTGGTACAATGTGG	CCTGATGGTTCACCCACTCC
Achn141271	GGGCTCTCGCGATATACGAC	TCTCCAACGTGAACGACGAG
Achn145641	TCCATGACATCACCAGCGTC	AGGAAGCATAACTGGCGTCC
Achn154591	ACGACATCACCACCGTCAAT	GCCGGTCCTTCACTTGGTAG
Achn172831	GGAGTGCTCCTCGGATTGTC	CCACTTTCTGCCACCGATCA
Achn207851	CTGTGGTACCCAGAATGCCT	ATGTCGTGGATGCTTGACCG
Achn212251	AAGGCCAACCGTGAATCTCC	AGGCAATCCGACATGGGTTC
Achn218481	AGAAGTACTTCATCCGGCAGC	GGAATTGCGTTCTTGGATTGG
Achn258201	CCCTGGCAACTTATCCCGAG	CTTGCGTCGCTCCTGATCTA
Achn365361	ACGACGGCAGTGATTATCGG	CTTGTCCTCGAACCTCGTCC
Achn371351	ATTGGAGGAGCATATCGCGG	TGGATGCTTGAACGTCTCCC
Acc00493（AcMYB10）	GCCATCACCACCATCCAACA	TTCCCCAAAATTGGCTCCTCA
Acc19563（AcbHLH42）	TCGTTTAGGGCCGAGTCTAC	TTGAGAGTAGTGCGTGTCGT
pSAK-Achn365361	ACTAGTGGATCCAAAGAATTCATGTATGGCGACGACGGC	AGAAGTACTCTCGAGAAGCTTTACATGGGATAGTTGAAGTTTTGGTACCC	过表达载体构建所用引物 Primers used for overexpression vector construction
pSAK-AcMYB10	ACTAGTGGATCCAAAGAATTCATGGAAAGTGTTACTTTAGGAGTGAGAAAGG	AGAAGTACTCTCGAGAAGCTTTAATCATCACCTAAGAGATCCCAAAGGTTAACA
pSAK-AcbHLH42	ACTAGTGGATCCAAAGAATTCATGGCGGCTCCCCCT	AGAAGTACTCTCGAGAAGCTTCAGGGTATGATTTGGTGTATTGCTCTCT
NtActin	AATGGAACTGGAATGGTCAAGGC	TGCCAGATCTTCTCCATGTCATCCCA	烟草RT-qPCR分析所用引物 Primers for real-time RT-qPCR analysis in tobacco
AcMYB88	ACGACGGCAGTGATTATCGG	CTTGTCCTCGAACCTCGTCC
AcMYB10	GCCATCACCACCATCCAACA	TTCCCCAAAATTGGCTCCTCA
AcbHLH42	TCGTTTAGGGCCGAGTCTAC	TTGAGAGTAGTGCGTGTCGT
NtC4H	ACAACCCGGATCACTGGAAG	ACTGTCCACCTTTCTCCGTG
NtCHS	TTGTTCGAGCTTGTCTCTGC	AGCCCAGGAACATCTTTGAG
NtPAL	TTACGCCCTTCGAACATCCC	GAGGCAAGTGCAAGCCTAGT
Nt4CL	ACTGCCGAAAGGTGTGATGT	AAATCGCTGCTCCGACTCTC
NtCHI	GTCAGGCCATTGAAAAGCTC	CTAATCGTCAATGCCCCAAC
NtF3H	CAAGGCATGTGTGGATATGG	TGTGTCGTTTCAGTCCAAGG
NtDFR	AACCAACAGTCAGGGGAATG	TTGGACATCGACAGTTCCAG
NtANS	TATCCCAAATGCCCCCAACC	ATCTTCTCCTTTGGCGGCTC
NtANR	TTCCATCTCTCATGGCTGGTC	ATATGGGCGCGACAAACATC

对照组 Control group	试验组 Experimental group
EV	OE10
EV	OE88
EV:OE10 : OE42（1:1:1）	OE88:OE10:OE42（1:1:1）
EV:OE10（1:1）	OE88:OE10（1:1）

对照组 Control group	试验组 Experimental group
EV	OE10
EV	OE88
EV:OE10 : OE42（1:1:1）	OE88:OE10:OE42（1:1:1）
EV:OE10（1:1）	OE88:OE10（1:1）

蛋白名称 Protein name	拟南芥同源蛋白 Homologous protein of Arabidopsis thaliana	氨基酸数 Number of amino acids	分子量 Molecular weight/Da	等电点 pI	带负电荷残基Asp+GIu	带正电荷残基 Arg+Lys	不稳定指数Instability	脂肪指数 Aliphatic index	亲水性 GRAVY	亚细胞定位Subcellular localization
Achn033551	AT1G49010.1	288	31 272.05	6.67	31	28	57.11	66.81	-0.54	细胞核Nucleus
Achn036201	AT2G38090.1	294	33 491.45	8.99	31	35	51.55	59.69	-0.79	细胞核Nucleus
Achn141271	AT2G38090.1	269	30 463.48	8.27	31	33	46.22	68.10	-0.58	细胞核Nucleus
Achn145641	AT1G49010.1	303	32 847.73	6.88	31	29	59.61	63.43	-0.61	细胞核Nucleus
Achn154591	AT2G38090.1	298	33 813.85	8.77	33	36	52.52	59.60	-0.81	细胞核Nucleus
Achn172831	AT5G58900.1	290	33 039.98	7.24	35	35	65.34	58.14	-0.87	细胞核Nucleus
Achn207851	AT5G08520.1	200	22 325.81	5.97	26	22	51.90	70.65	-0.62	细胞核Nucleus
Achn212251	AT2G38090.1	273	30 842.68	8.99	32	36	48.13	70.73	-0.69	细胞核Nucleus
Achn218481	AT5G58900.1	286	32 870.57	9.22	34	39	59.06	53.22	-1.02	细胞核Nucleus
Achn258201	AT5G08520.1	423	45 538.79	5.84	54	45	52.14	71.18	-0.54	细胞核Nucleus
Achn365361	AT5G04760.1	212	24 232.82	6.07	30	26	42.43	62.97	-0.81	细胞核Nucleus
Achn371351	AT5G08520.1	279	31 022.69	8.46	31	33	62.22	65.02	-0.67	细胞核Nucleus