百子莲MYB家族鉴定及蓝色形成关键基因功能分析

doi:10.16420/j.issn.0513-353x.2022-0226

摘要/Abstract

摘要：

利用生物信息鉴定百子莲MYB家族成员，挖掘并验证了调控百子莲蓝色花色形成的关键基因。以全长转录组测序数据作为参考序列，鉴定得到百子莲123个MYB家族成员，包括60个R2R3-MYB、4个3R-MYB、2个4R-MYB和57个1R-MYB基因。对调控类黄酮生物合成的主要亚家族R2R3-MYB进行分析发现，R2-和R3-repeats的序列较为保守，根据系统进化关系分为22个亚组（A1 ~ A22）。特殊的是，相较拟南芥的25个亚组，百子莲缺乏正向调控花色素苷合成的典型亚组S6。与花色形成相关的其他亚组有A18、A17和A16，其成员ApMYB4、ApMYB6、ApMYB7、ApMYB12和ApMYB111定位于细胞核，ApMYB123集中分布于核仁中。在蓝色百子莲花瓣着色过程中，ApMYB12显著上调表达，而ApMYB111显著下调表达。ApMYB4、ApMYB6和ApMYB7在蓝花各发育阶段的表达量均低于白花，预示这些基因可能是百子莲蓝色形成的负调控因子。ApMYB123在白花的开放前期大量表达，说明该基因可能在此阶段促进花瓣合成原花青素。在烟草中过表达ApMYB12，烟草花瓣颜色较野生型变浅，花色素苷含量减少54.55% ~ 62.50%，而黄酮醇含量提高125% ~ 170%，证实ApMYB12调控辅助色素黄酮醇的生物合成，从而影响蓝色的形成。

关键词: 百子莲, 转录因子, MYB家族, 蓝色花形成, 黄酮醇

Abstract:

MYB gene family members of Agapanthus praecox were identified by bioinformatics methods and the function of key genes that regulate blue flower coloration was verified. In total，123 ApMYBs were identified based on full-length transcriptome sequencing data，including 60 R2R3-MYBs，four 3R-MYBs，two 4R-MYBs and 57 1R-MYBs. Then，the R2R3-MYBs that regulate flavonoid biosynthesis were further analyzed. R2R3-MYBs were divided into 22 subgroups（A1-A22），together with A18（S4），A17（S5）and A16（S7）subgroups were all related to flower coloration，except for S6 subgroup. ApMYB4，ApMYB6，ApMYB7，ApMYB12，ApMYB111 and ApMYB123 were all localized in the nuclei. Whereas，ApMYB123 was especially concentrated in the nucleolus. During blue flower coloration，ApMYB12 was significantly up-regulated，while ApMYB111 was opposite. White flowers showed increased expression of ApMYB4，ApMYB6 and ApMYB7 compared to blue flowers，indicating them as the negative regulators of coloration. A specific expression peak of ApMYB123 was arisen in bloom stage of white flowers，suggesting biosynthesis of proanthocyanidins. Further ectopic expression of ApMYB12 in tobacco found that the petal color turned lighter than wild type plants. In addition，the anthocyanin content reduced by 54.55%-62.50% while the flavonols content increased by 125%-170%. These results demonstrated that ApMYB12 regulated the biosynthesis of auxiliary pigment flavonols，thereby regulating the coloration of blue flower.

Key words: Agapanthus praecox, transcription factor, MYB gene family, blue flower coloration, flavonol

中图分类号:

S682.32

贺威智, 雷伟奇, 郭祥鑫, 李蕊莲, 陈冠群. 百子莲MYB家族鉴定及蓝色形成关键基因功能分析[J]. 园艺学报, 2023, 50(6): 1255-1268.

HE Weizhi, LEI Weiqi, GUO Xiangxin, LI Ruilian, CHEN Guanqun. Identification of the MYB Gene Family and Functional Analysis of Key Genes Related to Blue Flower Coloration in Agapanthus praecox[J]. Acta Horticulturae Sinica, 2023, 50(6): 1255-1268.

图/表 9

表1 PCR引物列表

Table 1 Primer list for PCR

基因名称 Gene name	正向引物序列（5′-3′） Forward primer sequence	反向引物序列（5′-3′） Reverse primer sequence
ApActin	AACACCACCAGACTTATGT	GCCGTTGAACTTGATGAG
ApMYB4	CTACTGGAACACGCACATCAA	CTCTCGTCGGTAGAATCCTCAT
ApMYB6	AGTTGTCGGCTTCGTTGGA	TGTCTGTTCTTCCTGGTAGTCTT
ApMYB7	GACCTATCCATTAGCCTTCCTTATC	CTGTTGTTCTTGTCGCTGTCA
ApMYB12	GACCTCGACCAAGACTTGTTAC	ATCTAGCACCTTCTCCTCAACTT
ApMYB111	GGCACGGTATCAGCGGTAT	TTCTCATGCTCGTCTCTTGTTG
ApMYB123	CCTTATCATCCGACTCCACTCT	TTGCTGAGGTGACTGTTCCA
pHB-ApMYB4	CTCTCTCTCAAGCTTGGATCCATGGGTAGGTCTCCATGTTG	CATACTAGTGAGCTCCTGCAGAGCATTATACCCATCCTGTA
pHB-ApMYB6	CTCTCTCTCAAGCTTGGATCCATGGGTAGATCTCCTTGTTG	CATACTAGTGAGCTCCTGCAGCTTCATCTGAAAACTTCTAT
pHB-ApMYB7	CTCTCTCTCAAGCTTGGATCCATGGGAAGGTCCCCATGTTG	CATACTAGTGAGCTCCTGCAGTATCCATCCTTCCTCCAACG
pHB-ApMYB12	CTCTCTCTCAAGCTTGGATCCATGGGAAGGGCTCCATGTTG	CATACTAGTGAGCTCCTGCAGCAACACATCTGAAAGAAGCC
pHB-ApMYB111	CTCTCTCTCAAGCTTGGATCCATGGGGAGAGTTCCATGTTG	CATACTAGTGAGCTCCTGCAGTAATTTGCACCCACTCTCCC
pHB-ApMYB123	CTCTCTCTCAAGCTTGGATCCATGGGGAGAGCTCCATGCTG	CATACTAGTGAGCTCCTGCAGAATCAACAGAGGCTCTGCAG
NtPAL	ATTGAGGTCATCCGTTCTGC	ACCGTGTAACGCCTTGTTTC
Nt4CL	TCATTGACGAGGATGACGAG	TGGGATGGTTGAGAAGAAGG
NtCHS	TTGTTCGAGCTTGTCTCTGC	AGCCCAGGAACATCTTTGAG
NtCHI	GTCAGGCCATTGAAAAGCTC	CTAATCGTCAATGCCCCAAC
NtFLS	TTTGGCACTTGGTGTTGTGG	ACTTGACATCATACCAATGG
NtDFR	AACCAACAGTCAGGGGAATG	TTGGACATCGACAGTTCCAG
NtUFGT	ATGTTGAAGGGCTAAAAGAAAGAGC	CAAGTCCCAGCTGATACATATTCCC
NtGAPDH	GGTGTCCACAGACTTCGTGG	GACTCCTCACAGCAGCACCA

图1 百子莲（A）与拟南芥（S）R2R3-MYB的系统进化树

Fig. 1 Phylogenetic tree of R2R3-MYB proteins between Agapanthus praecox（A）and Arabidopsis thaliana（S）

图2 百子莲R2R3-MYB结构域序列分析 Bits值表示序列中每个氨基酸位点的保守程度，星号代表MYB结构域中的保守氨基酸残基色氨酸（W）。

Fig. 2 Consensus sequence of R2R3-MYB domains in Agapanthus praecox The bit score of Y-axis indicated the conservation rate for each X-axis amino acid position in the sequence. Asterisks indicate the conserved residues Trp（W）in the MYB domain.

图3 蓝花‘蓝色大花’与白花‘新娘花束’百子莲花瓣发育过程中的花色素苷含量 S1：现蕾期；S2：花蕾伸长期；S3：开放前期。不同小写字母表示花色素苷含量差异显著（P < 0.05）。

Fig. 3 The anthocyanin contents during flower development in blue-flowered‘Big Blue’and white-flowered‘Bridal Bouquet’ S1：Bud emergence period；S2：Bud elongation period；S3：Pre-opening period. The different lowercase letters respectively indicate the significant difference in the anthocyanin content（P < 0.05）.

图4 蓝花‘蓝色大花’与白花‘新娘花束’百子莲花瓣着色过程中类黄酮合成相关MYB基因表达热图（A）和蛋白保守基序（B） S1：现蕾期；S2：花蕾伸长期；S3：开放前期。

Fig. 4 Heat map of expression（A）and protein conserved motifs（B）of MYB genes related to flavonoid biosynthesis during flower coloration in blue-flowered‘Big Blue’and white-flowered‘Bridal Bouquet’ S1：Bud emergence period；S2：Bud elongation period；S3：Pre-opening period.

图5 类黄酮合成调控相关MYB蛋白的亚细胞定位（标尺 = 50 μm）

Fig. 5 The subcellular localization of MYB proteins related to flavonoid biosynthesis（Bar = 50 μm）

图6 烟草转ApMYB12株系（7#、8#）及野生型（WT）的花色表型、花瓣和叶片总黄酮醇和总花色素苷含量 **代表野生型与转基因株系间差异极显著（P < 0.01）。

Fig. 6 Flower color phenotypes, flavonol and anthocyanin content in petal and leaf of ApMYB12-transgenic tobacco lines（7#，8#）and wild type ** represents the significant difference between wild-type and transgenic lines（P < 0.01）.

图7 野生型和转基因烟草花瓣中类黄酮合成通路

Fig. 7 The flavonoid biosynthesis pathway of ApMYB12-transgenic tobacco lines and wild type

图8 烟草转ApMYB12株系（7#、8#）及野生型（WT）中类黄酮合成途径关键基因的表达量不同小写字母表示野生型和转基因株系间具有显著差异（P < 0.05）。

Fig. 8 The expression of key genes in the flavonoid biosynthesis pathway of of ApMYB12-transgenic tobacco lines（7#，8#）and wild type Different lowercase letters indicate significant differences between wild-type and transgenic lines（P < 0.05）.

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