园艺学报 ›› 2021, Vol. 48 ›› Issue (3): 465-476.doi: 10.16420/j.issn.0513-353x.2020-0468
朱自果1, 张庆田1, 韩真1, 李勃1, 李国栋2, 李秀杰1,*()
收稿日期:
2020-11-24
出版日期:
2021-03-25
发布日期:
2021-04-02
通讯作者:
李秀杰
E-mail:lixiujie-2007@163.com
基金资助:
ZHU Ziguo1, ZHANG Qingtian1, HAN Zhen1, LI Bo1, LI Guodong2, LI Xiujie1,*()
Received:
2020-11-24
Online:
2021-03-25
Published:
2021-04-02
Contact:
LI Xiujie
E-mail:lixiujie-2007@163.com
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。
中图分类号:
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.
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 |
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.
编号 ID | 代谢物 Metabolites | 相对含量 Relative concentration | OE1/WT | |
---|---|---|---|---|
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 |
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 | |
---|---|---|---|---|
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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