欧洲葡萄VvMYB6正向调控花青素合成

doi:10.16420/j.issn.0513-353x.2020-0468

摘要/Abstract

摘要：

从欧洲葡萄‘粉红亚都蜜’中克隆到1个MYB转录因子基因VvMYB6。VvMYB6蛋白定位在细胞核,其N端包含1个保守的R2R3结构域。在葡萄中,VvMYB6主要在在根、花器官及果实发育早期表达。在烟草中异位表达VvMYB6,显著促进花瓣和雄蕊中花青素的积累;代谢组分析发现,相比野生型,转基因植株花中累积高含量的飞燕草色素和矢车菊色素。转基因烟草植株中查尔酮合酶（CHS）、查尔酮异构酶（CHI）、黄烷酮4-还原酶（DFR）、花青素合酶（ANS）和UDP葡萄糖-类黄酮-O-葡萄糖基转移酶（UFGT）基因表达显著上调。结果表明VvMYB6正向调控花青素合成。

关键词: 葡萄, MYB转录因子, 花青素

Abstract:

Here,we demonstrated that VvMYB6,an R2R3-MYB transcription factor,may function as an activator of anthocyanin biosynthesis in grapevine（Vitis vinifera L.）. The VvMYB6 protein contains a conserved R2R3 domain at its N-terminal and localized to the nucleus. Expression studies in grapevine showed that VvMYB6 were strongly expressed in roots,flowers,and berry development of early stages. Ectopic expression of VvMYB6 in tobacco caused the accumulation of anthocyanins in petals and stamens. Metabolomics analysis showed that VvMYB6 expression also caused a marked accumulation of several phenolic compounds,including delphinidin and cyanidin. Expression analysis revealed that several anthocyanin biosynthesis structural genes,including chalcone synthease（CHS）,chalcone isomerase（CHI）,dihydroflavonol 4-reductase（DFR）,anthocyanidin synthase（ANS）and UDP glucose flavonoid 3-O-glucosyl transferase（UFGT）,were strongly up-regulated in transgenic plants. These findings suggest that VvMYB6 plays a key role in anthocyanin biosynthesis.

Key words: grapevine, MYB transcription factor, anthocyanin

中图分类号:

S663.1

朱自果, 张庆田, 韩真, 李勃, 李国栋, 李秀杰. 欧洲葡萄VvMYB6正向调控花青素合成[J]. 园艺学报, 2021, 48(3): 465-476.

ZHU Ziguo, ZHANG Qingtian, HAN Zhen, LI Bo, LI Guodong, LI Xiujie. VvMYB6,an R2R3-MYB Transcription Factor,is Involved in Anthocyanin Biosynthesis of Grapevine[J]. Acta Horticulturae Sinica, 2021, 48(3): 465-476.

图/表 11

表1 定量引物

Table 1 Sequence of the primers in qPCR experiment

基因名称 Gene name	登录号 Accession number	正向引物序列（5′-3′） Forward primer	反向引物序列（3′-5′） Reverse primer
VvMYB6	MN125488	CTTATCATCCGGCTTCGTTCCCT	TTTGCCCGTGTGCTTCAATCTCT
VvActin	XP_002282516	CCTCAACCCCAAGGCCAACAGA	ACCATCACCAGAATCCAGCACA
NtCHI	X75963	GTCAGGCCATTGAAAAGCTC	CTAATCGTCAATGCCCCAAC
NtCHS	AB066274	TTGTTCGAGCTTGTCTCTGC	AGCCCAGGAACATCTTTGAG
NtFLS	AB086055	GAACTTGAAGGGAAAAGGGG	TCCCTGTAGGAGGGAGGATT
NtDFR	NM_001281215	AACCAACAGTCAGGGGAATG	TTGGACATCGACAGTTCCAG
NtANR1	NM_001280956	CATTTGACTTTCCCAAACGC	ATTGGGCTTTTGAGTTGTGC
NtANR2	XP_016512400	TGTTCCCACTTGGGATGATA	TGCACCTATACTCTGTTAGTGGC
NtANS	JQ866631	TGGCGTTGAAGCTCATACTG	GGAATTAGGCACACACTTTGC
NtUFGT	AF000371	GAGTGCATTGGATGCCTTTT	CCAGCTCCATTAGGTCCTTG
NtPAL	EF192469	CCTTTATCCTACATTGCTGGTTT	TCGGGCTTTCCGTTCATTACTTC
NtActin	XM_016618072	AATGGAACTGGAATGGTCAAGGC	TGCCAGATCTTCTCCATGTCATCCCA

图1 VvMYB6基因在葡萄基因组中的定位（A）和VvMYB6蛋白进化树分析（B）

Fig. 1 The genomic sequence of VvMYB6 which is mapped to Vitis vinifera‘Pinot Noir’clone P40024 genome（A）and phylogenetic tree（B）of R2R3-MYB transcription factors from grapevine and other species

图2 VvMYB6蛋白与其他MYB蛋白的同源性分析

Fig. 2 Alignment of the deduced amino acid sequence of VvMYB6 with other MYB proteins

图3 VvMYB6蛋白的亚细胞定位

Fig. 3 Subcelluar localization of VvMYB6 protein

图4 VvMYB6基因的组织表达分析

Fig. 4 Expression analysis of VvMYB6 in different tissue

图5 VvMYB6基因启动子的生物信息（A）和转基因烟草幼苗、花和种子的GUS染色（B）

Fig. 5 Promoter analysis of VvMYB6 gene（A）and GUS staining on 10-day-old tobacco seedling,flowers at different development and seeds（B）

图6 转基因烟草（OE1、OE3和OE5）和野生型烟草的花表型 i:花瓣;ii:雄蕊;iii:花筒。

Fig. 6 Phenotypes of transgenic tobacco（OE1,OE3 and OE5）and wild-type tobacco flowers i:Petal;ii:Stamen;iii:Corolla tube.

图7 转基因株系VvMYB6的表达水平

Fig. 7 Expression level of VvMYB6 in transgenic tobacco lines

图8 VvMYB6促进转基因烟草花青素的积累 ANOVA and Tukey’s test,与对照比较,* α = 0.05。

Fig. 8 Anthocyanin accumulation in VvMYB6 transgenic plants Significant difference from the wild type was confirmed by ANOVA and Tukey’s test. *α = 0.05.

表2 转基因烟草OE1株系花中代谢物的相对含量

Table 2 Relative concentration and fold-changes in the levels of major metabolites in flowers of transgenic OE1 line

编号 ID	代谢物 Metabolites	相对含量 Relative concentration		OE1/WT
编号 ID	代谢物 Metabolites	OE1	WT
M466T139	飞燕草色素 Delphinidin	36 999.82 ± 1 938.03	16 293.89 ± 822.94	2.27
M488T142	飞燕草色素 Delphinidin	7 152.78 ± 666.11	1 809.76 ± 235.23	3.95
M552T143	飞燕草色素 Delphinidin	2 705.68 ± 253.64	964.45 ± 107.88	2.81
M597T60	飞燕草色素 Delphinidin	6 892.49 ± 643.20	2 622.56 ± 352.73	2.63
M604T134	矢车菊色素 Cyanidin	25 610.01 ± 955.04	8 350.96 ± 311.87	3.07
M612T139	矢车菊色素 Cyanidin	121 588.30 ± 14 228.53	55 138.26 ± 3 964.54	2.21
M628T137	飞燕草色素 Delphinidin	8 934.53 ± 350.41	3 835.04 ± 346.59	2.33
M637T55	矢车菊色素 Cyanidin	625.84 ± 97.10	261.51 ± 34.42	2.39
M774T133	飞燕草色素 Delphinidin	15 256.59 ± 410.53	4 467.30 ± 148.95	3.42
M790T151	飞燕草色素 Delphinidin	1 220.32 ± 72.70	437.53 ± 62.86	2.79
M796T133	飞燕草色素 Delphinidin	10 307.65 ± 399.36	3 850.49 ± 247.85	2.68

图9 转基因烟草花瓣（A）和雄蕊（B）中花青素合成相关基因的表达 ANOVA and Tukey’s test,与对照比较,* α = 0.05。

Fig. 9 Expression analysis of anthocyanin biosynthetic genes in petal（A）and stamen（B）from three VvMYB6 transgenic lines and wild-type lines Significant difference from the wild type was confirmed by ANOVA and Tukey’s test. *α = 0.05.

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