Anthocyanin is a class of water-soluble natural pigment that exists widely in nature, and it is an important secondary metabolite in plants, which could make plant leaves and petals show colorful colors (Fu et al., 2018). The accumulation of anthocyanins could help plants enhance free radical scavenging and antioxidant, resist environmental stresses such as low temperature and drought, and protect the tissues for photosynthesis (Butelli et al., 2012; Kim et al., 2017). Meanwhile, edible foods rich in anthocyanins have many biological health care functions, such as fighting cancer and improving cardiovascular (Puiggròs et al., 2014). Therefore, plant resources rich in anthocyanins are highly valued by breeders, and have great application prospects in the breeding of ornamental, vegetable and stress resistant varieties.

The transcription of the key enzyme genes for anthocyanin synthesis is mainly regulated by the MBW complex, including MYB, bHLH and WD40 transcription factors. Among them, MYB transcription factor is the most numerous, which plays an important role in regulating the synthesis and accumulation of anthocyanins (Dubos et al., 2010; Xu et al., 2014; Yao et al., 2017). The proteins encoded by the PAP1 (Production of Anthocyanin Pigment 1) gene belongs to R2R3 MYB transcription factor, which can regulate the expressions of DFR, ANS/LDOX, TT19, TT8, GL3, EGL3 and other genes to promote the synthesis and accumulation of anthocyanins (Maier et al., 2013; Yan et al., 2019). In Brassica oleracea (CC, 2n=18), the up-regulated expression of BoPAP1 is an important reason for the appearance of purple leaf traits. However, it is not completely clear whether there is a similar mechanism in other Brassica species.

Brassica juncea (AABB, 2n=36) belongs to Brassica of Brassicaceae, which is an important raw material of oil and vegetable crop in China, with abundant variation resources (Yang et al., 2016; Yang et al., 2018). Some genes related to the color of the seed coat of Brassica juncea have been cloned, while there are fewer studies on cloning and expression of gene related to leaf anthocyanin synthesis (Yan et al., 2007; Yan et al., 2011). Brassica rapa (AA, 2n=20), as the ancestor of Brassica juncea, provides a good sequence reference for homologous cloning because its anthocyanin synthesis pathway is relatively clear (Guo et al., 2014).

The purpose of this study is to clone the PAP1 gene from Brassica juncea with different leaf colors through the method of homologous cloning. And we also want to use bioinformatics methods to predict the structure and function of the protein encoded by the PAP1 gene, to compare its sequence and expression differences among leaves with different colors, and to explore the relationship between PAP1 gene and leaf color. This study provides a reference for exploring the function of PAP1 gene and the formation mechanism of different leaf color of Brassica juncea.

1 Results and Analysis

1.1 The phenotype of Brassica juncea with different leaf colors

The whole leaves of B. juncea (lv) are green. B. juncea (hong) is lavender overall and the purple radiates from the edge of the leaf to the middle, while the veins are green. The leaves and veins of B. juncea (zi) are dark purple. Their phenotypes are obviously different (Figure 1A). Moreover, it was found that the anthocyanin content from high to low is B. juncea (zi), B. juncea (hong) and B. juncea (lv) by measuring. The anthocyanin content in B. juncea (zi) is twice that of B. juncea (hong) and 9 times that of B. juncea (lv). Their anthocyanin content is also obviously different (p<0.01) (Figure 1B). Therefore, we could know the phenotype of Brassica juncea with different leaf colors related to anthocyanin content.

Figure 1 Comparison of leaf phenotype and anthocyanin content of Brassica juncea with different leaf colors Note: A: The leaf types from left to right are Brassica juncea of purple, red and green; B: Different capital letters indicate significant differences at 0.01 level

1.2 Cloning of PAP1 gene in Brassica juncea

Brassica rapa and Brassica juncea are both belongs to Brassica, and Brassica rapa is the ancestor of Brassica juncea, so they two have high homology. We used the DNA of Brassica juncea of purple, red and green as templates and designed homologous primers for PCR amplification by using the BraPAP1 gene sequence of Brassica rapa. After agarose gel electrophoresis, we could see base pair is like a single strip with higher brightness, the number of which is 1 546, 1 669, 1 664, 1 348, 1 598 (Figure 2). Then five PAP1 gene sequences in Brassica juncea with different leaf colors were successfully cloned.

Figure 2 Amplification results of primer PAP1.2 in purple leaf Note: M: DL2501 DNA marker; (zi-2): PCR product of purple leaf

1.3 Sequence analysis of PAP1 gene in Brassica juncea

Through homologous cloning, the 5 sequences with higher homology were labeled as B.juncea (zi), B.juncea (zi-2), B.juncea (lv), and B.juncea (lv-2), B.juncea (hong). The GenBank accession numbers of the cloned PAP1 genes are MT210230, MT210231, MT210232, MT210233 and MT210234. The sequencing results showed that the PAP1 gene length of Brassica juncea is from 1 348 bp to 1 669 bp, and the CDS length is from 744 bp to 753 bp. It also showed the gene sequence includes 3 exons and 2 introns, and the number of encoded amino acids is from 247 to 250 (Table 1).

Table 1 Basic information of PAP1 gene in different leaf colors Brassica juncea

1.4 Bioinformatic analysis of PAP1 gene

Bioinformatic analysis showed that the theoretical molecular weight of the protein encoded by the PAP1 gene is from 27 941.7 to 28 613.4 and the theoretical isoelectric point is from 8.75 to 9.17, which proved that the protein is a basic protein. Using the online website ProtParam to analyze the proportion of amino acids in the PAP1 protein, we got the result that Leucine (Leu) has the highest amino acid content, which accounts for more than 10%. Using the ProtScale website (https://web.expasy.org/cgi-bin/protscale/protscale.pl/) to analyze the hydrophilicity and hydrophobicity of the PAP1 protein, we found that PAP1 has no typical hydrophobic region, which showed PAP1 protein is hydrophilic.

The secondary structure of PAP1 gene is mainly composed of α-helix (32.79%~38.96%), extended strand (9.31%~12.96%), β turn (8.91%~12.80%) and random coil (38.80%~45.34%) (Table 2). Using SWISS-MODEL (https://swissmodel.expasy.org/) software, we constructed tertiary structure of PAP1 protein (Figure 3).

Table 2 Physicochemical properties of PAP1 protein in Brassica juncea with different leaf colors

Figure 3 Prediction of tertiary structure of PAP1 protein in Brassica juncea

Using the SMART website (http://smart.embl-heidelberg.de/) to predict and analyze the conserved domain of PAP1 protein and using the online website Plant-mPLoc to predict and analyze the subcellular location of PAP1 protein, we got the result that the conserved domain of PAP1 protein includes two typical SANT binding domains (MYB binding domains), which respectively locates from the 9^th to 59^th and from 62^nd to 110^th of amino acid sequence (Figure 4). Subcellular location predicted that the protein is located in the nucleus. It was further proved that the cloned gene is the PAP1 gene of R2R3 MYB transcription factor family.

Figure 4 Comparison of amino acid sequences encoded by PAP1 gene in Brassica juncea with different leaf colors Note: The background color black indicates the same amino acid, and there is a difference in the amino acid sequence of the unlabeled black; the black box indicates the conserved SANT domain

1.5 Phylogenetic analysis of the coding sequence of PAP1 gene

Putting the PAP1 protein sequence in Brassica juncea into the NCBI database for Blast search and comparison, and selecting the amino acid sequence of the relative plant with the highest homology to PAP1 for multiple sequence alignment and analysis, and then using MEGA v.7 software to build a phylogenetic tree, we got the result that the amino acid sequence of PAP1 in Brassica juncea has the highest homology with the amino acid sequence of B.rapa. The homology is from 92% to 100%. Among them, B.juncea (zi), B.juncea (lv) and B.juncea (hong) have high homology with Bra004162 in the Brassica database. While B.juncea (zi-2) and B.juncea (lv-2) have high homology with Bra001917 and Bra039763, respectively. It showed that the PAP1 protein is highly conservative in evolution. The amino acid sequence of PAP1 in Brassica juncea also has high homology with other relative plants of the same genus such as B.napus, B.rapa subsp. rapa, B.oleracea var. botrytis and so on. The homology is from 89% to 98%. The phylogenetic tree showed that the model plant A.thaliana is distributed outside the phylogenetic tree, indicating that the PAP1 protein in A.thaliana is relatively distant from the PAP1 protein in Brassica juncea (Figure 5).

Figure 5 Phylogenetic analysis between PAP1 protein of Brassica juncea and other relative plants

1.6 Comparison of PAP1 gene sequence in Brassica juncea with different leaf colors

It was found that there are certain differences among the PAP1 gene sequence in B.juncea(zi), B.juncea(lv) and B.juncea(hong) by homology comparison analysis. The differences between PAP1 gene sequence in B.juncea(zi) and that in B.juncea(hong), which are both homologous to Bra004162, are mainly concentrated in the intron region (Figure 6). But the coding sequence and amino acid sequence of the two are exactly the same (Figure 4). The sequence differences between B.juncea(zi)/B.juncea(hong) and B.juncea(lv) also exist in the coding region. Although B.juncea(zi)/B.juncea(hong) and B.juncea(lv) encode 247 amino acids, there are 22 differences in the amino acid sequence. Therefore, it was speculated that the difference of PAP1 gene sequence in Brassica juncea may be the reason for difference of leaf color.

Figure 6 CDS sequences comparison of different leaf colors Brassica juncea PAP1 gene that homologous to Bra004162 of B.rapa

1.7 Expression analysis of PAP1 gene and its regulatory genes

Extracting RNA from young leaves of B.juncea (zi) and B.juncea (lv) at the three-leaf stage, and using quantitative PCR to analyze the expressions of PAP1 gene and its downstream genes DFR, TT19, TT8 and so on, we got the result that the expressions of PAP1 gene and its downstream genes DFR, TT19, TT8, GL3 and EGL in B.juncea (lv) is lower than that in B.juncea (zi) (p<0.05), and the expressions of DFR and TT19 in B.juncea (zi) is 5 to 7 times higher than that in B.juncea (lv) (Figure 7). It was known that the transcription factor encoded by the PAP1 gene controls the expressions of downstream genes and affects the synthesis of anthocyanins. Furthermore, it was also known that the expression difference of each gene is correlated with the leaf color of B.juncea (zi) and B.juncea (lv). Therefore, it was speculated that different leaf colors may be caused by different expressions of genes.

Figure 7 Comparison of the expression levels of PAP1 and its downstream regulatory genes in Brassica juncea with different leaf colors

Through the cloning and expression analysis of the PAP1 gene in Brassica juncea with different leaf colors, it was found that the evolution of the PAP1 gene is extremely conservative. Moreover, there are different sequences and expressions of PAP1 gene in B.juncea (zi) and B.juncea (lv). The expressions of PAP1 and its downstream genes are significantly down-regulated in B.juncea (lv). In a word, it was speculated that the above differences may be the reason for appearance of different leaf colors of Brassica juncea.

2 Discussion

Anthocyanin is important secondary metabolites in plants. Brassica juncea is rich in genetic resources and crops with high anthocyanin content have great application prospects in ornamental, vegetable and stress resistance. Jeon et al. (2018) conducted a comprehensive analysis of the transcriptome and metabolome of B. oleracea (lv) and B. oleracea (hong). It was found that B. oleracea (hong) contains more anthocyanins than B. oleracea (lv) and the expression of anthocyanin synthesis gene is positively correlated with anthocyanin content. Moreover, it was found that the anthocyanin content in B. juncea (zi) is significantly higher than that in B. juncea (hong) and B. juncea (lv), and the expressions of related genes involved in the anthocyanin synthesis in B. juncea (zi) is higher than that in B. juncea (lv), indicating that anthocyanin is the main reason for leaf color changes.

The anthocyanin synthesis involves several structural genes and regulatory genes, among which, MYB is the most important and most numerous transcriptional regulatory factors. According to the number of its structural domains, it can be divided into four categories, namely 1R-MYB, R2R3-MYB, 3R -MYB and 4R-MYB. The expressions of R2R3 MYB transcription factors PAP1 and PAP2 can increase the expressions of structural genes in anthocyanin biosynthesis. In recent years, a large number of studies have shown that PAP1 is a key transcription factor of MYB that regulates anthocyanin synthesis. The expression of anthocyanin was up regulated by binding to the promoter of the target gene, thus promoting the accumulation of anthocyanins (Borevitz et al., 2000). Liu et al. (2017) cloned the Arabidopsis thaliana AtPAP1 gene from the Arabidopsis thaliana inflorescence. They constructed a gene expression vector and transferred it into tobacco for heterologous gene expression. The results showed that the tobacco plants show different colors, indicating that the overexpression of PAP1 gene has an effect on the color of tobacco plants. In this study, five PAP1 gene sequences of Brassica juncea with different leaf colors were cloned by using homologous cloning technology. Through structural domain analysis, it was found that the PAP1 gene in Brassica juncea contains two conserved domains, namely the MYB binding domain, which can be combined with the target gene promoter to induce the expressions of downstream genes, and its structure is consistent with the predicted function, which showed that this gene plays a role in anthocyanin biosynthesis. The homologous alignment of the gene sequence showed that the PAP1 gene has high homology with the homologous sequence of Brassica rapa. Phylogenetic tree analysis showed that the PAP1 protein also has high homology with other related plants of the Brassica genus, and it is relatively distant from A.thaliana, which indicated that the gene is conservative in evolution. Compared with A.thaliana, Brassica experiences genome triploidization, so the PAP1 gene also has multiple copies in Brassica juncea (Wang et al., 2011; Yang et al., 2016). In this study, five sequences were cloned, of which three were homologous to Bra004162, one was homologous to Bra001917 and one was homologous to Bra039763. Among them, the amino acid sequence of PAP1 in B.juncea (zi) and B.juncea (hong) is exactly the same, but there are 22 differences from the amino acid sequence of B.juncea (lv). Gene expression analysis showed that PAP1 gene and its downstream regulatory genes are down-regulated in B.juncea (lv), leading to lower anthocyanin content than B.juncea (zi). The up-regulated expression of BoPAP1 in Brassica oleracea is an important reason for the appearance of purple leaf (Zhang et al., 2012). Therefore, we speculated that different sequences and expressions of PAP1 gene may lead to the differences of leaf color in Brassica juncea. The specific mechanism of the appearance of color difference remains to be further studied.

3 Materials and Methods

3.1 Materials

Brassica juncea with different leaf colors: including B.juncea (lv) (green leaf), B.juncea (hong) (lavender leaf), B.juncea (zi) (purple leaf). The leaves were provided by the Yan Mingli Laboratory of Hunan Science and Technology University and grew in the Biological Park of Hunan Science and Technology University. Primer synthesis, cloning and sequencing were provided by Tianyi Huiyuan Biotechnology Company. Plant DNA recovery and RNA extraction kits were purchased from Tiangen Biochemical Technology Co., Ltd. pMD18-T vector was purchased from TaKaRa company. E. coli DH5α strain, PCR and other reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd.

3.2 Determination of anthocyanin content in different leaf colors

Referring to Li et al. (2016), we took fresh leaves and dried them, then extracted 0.1 g of dried leaves and ground them into powder to extract anthocyanin. Using a UV spectrophotometer (Agilent Technologies Cary60 UV-Vis) to measure the absorbance at 530 nm, we calculated the total anthocyanin content in leaves by using the formula: anthocyanin content (mg/g)=A530×N×10×98.2-1. 

3.3 Cloning of PAP1 gene

The young leaves of Brassica juncea with different leaf colors (purple, red, green) at the three-leaf stage were collected, and the leaf DNA was extracted by the CTAB method. We took the published PAP1 gene sequence (Bra039763; Bra001917; Bra004162) of Brassica rapa in the Brassica database (http://brassicadb.org/brad/) as a template, the primers PAP1.1 (Bra039763 homology) PAP1.2 (Bra001917 homology) PAP1.3 (Bra004162 homology) were designed online through the NCBI-BLAST website. The annealing temperature of the primer was set at 55℃ for 35 cycles. The PCR products were detected by 1% agarose gel electrophoresis, and the results were observed with a UV gel imaging analyzer. After the PCR product was purified by agarose gel DNA recovery kit, it was connected to the pMD18-T vector to transform the competent cells of E. coli DH5α. After resuscitation, plating and colony culture, we marked and picked the scattered and smooth single colonies, and used M13 universal primers for bacterial liquid PCR, the primer sequence (Table 3). After the positive clones were screened out, the positive clones were sent to the company for sequencing.

Table 3 Primer names and sequences for gene cloning and expression analysis

3.4 Bioinformatic analysis of PAP1 gene

We used DNAMAN v.8 software to splice the sequence. According to the Brassica database, we inferred the exon and intron regions of the sequence and translated it into a protein sequence to predict its isoelectric point. The online website ProtParam (https://web.expasy.org/protparam/) was used to analyze the amino acid composition and hydrophobicity of the protein sequence encoded by the PAP1 gene. The MEGA7.0.14 software was used to compare the PAP1 protein sequence of Brassica juncea with PAP1 homologous sequences of other types of plants, and constructed a phylogenetic tree. Finally, we used PRABI, SWISS-MODEL, SMART and other websites to speculate on the secondary structure, tertiary structure and conserved domains of the protein encoded by the PAP1 gene.

3.5 Expression analysis of PAP1 gene

Taking the young leaves of B.juncea (lv) and B.juncea (zi) at the three-leaf stage, extracting the total RNA by using TRIzol kit, and removing impurity DNA by using RQ1 DNase (promega), we got purified RNA. Then the quality and concentration of purified RNA were measured under the absorbance of a UV spectrophotometer A260/A280. And its integrity was check by agarose gel electrophoresis. The obtained RNA was reverse transcribed with the ReverAid^TM First Strand cDNA Synthesis kit to obtain cDNAs in B.juncea (lv) and B.juncea (zi). Primer software was used to design suitable primers (Table 3), and the Actin gene was used as an internal reference primer for qRT-PCR amplification to perform gene expression analysis.

Authors’ contributions

HD designed and executed the experiments. And she completed data analysis and wrote the first draft of the manuscript. ZWP, LW CJL and HP participated in designing the experiments and analyzing the results. ZDW conceived the project and was responsible for the project. And he directed the experiments, data analysis, paper writing and revision; YML participated in directing the experiment, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This research was jointly funded by the Natural Science Foundation of China Youth Project (31701462), the Scientific Research Project of Hunan Education Department (18C0305), and the General Project of Graduate Research and Innovation of Hunan Province (CX20201007).

References

Borevitz J.O., Xia Y., Blount J., Dixon R.A., and Lamb C., 2000, Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis, Plant Cell, 12(12): 2383-2393

https://doi.org/10.1105/tpc.12.12.2383
PMid:11148285 PMCid:PMC102225

Butelli E., Licciardello C., Zhang Y., Liu J., Mackay S., Bailey P., Reforgiato-Recupero G., and Martin C., 2012, Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges, Plant Cell, 24(3): 1242-1255

https://doi.org/10.1105/tpc.111.095232
PMid:22427337 PMCid:PMC3336134

Dubos C., Stracke R., Grotewold E., Weisshaar B., Martin C., and Lepiniec L., 2010, MYB transcription factors in arabidopsis, Trends in Plant Science, 15(10): 0-581

https://doi.org/10.1016/j.tplants.2010.06.005
PMid:20674465

Fu W.Q., Chen D.Z., Pan Q., Li F.F., Zhao Z.G., Ge X.H., and Li Z.Y., 2018, Production of red-flowered oilseed rape via the ectopic expression of Orychophragmus violaceus OvPAP2, Plant Biotechnol. J., 16(2): 367-380

https://doi.org/10.1111/pbi.12777
PMid:28640973 PMCid:PMC5787836

Guo N., Cheng F., Wu J., Liu B., Zheng S.N., Liang J.L., and Wang X.W., 2014, Anthocyanin biosynthetic genes in Brassica rapa, BMC Genomics, 15(1): 426

https://doi.org/10.1186/1471-2164-15-426
PMid:24893600 PMCid:PMC4072887

Jeon J., Kim J.K., Kim H., Kim Y.J., Park Y.J., Kim S.J., Kim C., and Park S.U., 2018, Transcriptome analysis and metabolic profiling of green and red kale (Brassica oleracea var. acephala) seedlings, Food Chemistry, 241: 7-13

https://doi.org/10.1016/j.foodchem.2017.08.067
PMid:28958560

Kim J., Lee W.J., Vu T.T., Jeong C.Y., Hong S.W., and Lee H., 2017, High accumulation of anthocyanins via the ectopic expression of AtDFR confers significant salt stress tolerance in Brassica napus L, Plant Cell Reports, 36(8): 1215-1224

https://doi.org/10.1007/s00299-017-2147-7
PMid:28444442

Li H.B., Zhu L.X., Yuan G.G., Heng S.P., Yi B., Ma C.Z., Shen J.X., Tu J.X., Fu T.D., and Wen J., 2016, Fine mapping and candidate gene analysis of an anthocyanin-rich gene, BnaA.PL1, conferring purple leaves in Brassica napus L., Mol. Genet. Genomics., 291(4): 1523-1534

https://doi.org/10.1007/s00438-016-1199-7
PMid:27003438

Liu Y., Zhen T.C., Dai L.J., Liu C.X., Wang Q.N., and Qu G.Z., 2017, Construction of plant expression vector and genetic transformation analysis of Arabidopsis thaliana, Zhiwu Shengli Xuebao (Plant Physiology Journal), 53(7): 1199-1207)

Maier A., Schrader A., Kokkelink L., Falke C., Welter B., Iniesto E., Rubio V., Uhrig J.F., Hulskamp M., and Hoecker U., 2013, Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis, Plant J., 74(4): 638-651

https://doi.org/10.1111/tpj.12153
PMid:23425305

Puiggròs F., Salvadó M.J., Blade C., and Arola L., 2014, Differential modulation of apoptotic processes by proanthocyanidins as a dietary strategy for delaying chronic pathologies, Crit. Rev. Food Sci., 54(3): 277-291

https://doi.org/10.1080/10408398.2011.565456
PMid:24188302

Wang X.W., Wang H.Z., Wang J., Sun R., Wu J., Liu S.Y., Bai Y.Q., Mun J.H., Bancroft I., Cheng F., Huang S.W., Li X.X., Hua W., Wang J.Y., Wang X.Y., Freeling M., Pires J. C., Paterson A. H., Chalhoub B., Wang B., Hayward A., Sharpe A.G., Park B.S., Weisshaar B., Liu B.H., Li B., Liu B., Tong C.B., Song C., Duran C., Peng C.F., Geng C.Y., Koh C., Lin C.Y., Edwards D., Mu D., Shen D., Soumpourou E., Li F., Fraser F., Conant G., Lassalle G., King G.J., Bonnema G., Tang H.B., Wang H.P., Belcram H., Zhou H.L., Hirakawa H., Abe H., Guo H., Wang H., Jin H.Z., Parkin I. A., Batley J., Kim J. S., Just J., Li J., Xu J.H., Deng J., Kim J. A., Li J.W., Yu J.Y., Meng J.L., Wang J.P., Min J.M., Poulain J., Wang J., Hatakeyama K., Wu K., Wang L., Fang L., Trick M., Links M.G., Zhao M.X., Jin M., Ramchiary N., Drou N., Berkman P.J., Cai Q.L., Huang Q.F., Li R.Q., Tabata S., Cheng S.F., Zhang S.J., Zhang S., Huang S.M., Sato S., Sun S.L., Kwon S. J., Choi S.R., Lee T.H., Fan W., Zhao X., Tan X., Xu X., Wang Y., Qiu Y., Yin Y., Li Y.R., Du Y.C., Liao Y.C., Lim Y., Narusaka Y., Wang Y.P., Wang Z.Y., Li Z.Y., Wang Z.W., Xiong Z.Y., Zhang Z.H., and Brassica rapa genome sequencing project, consortium, 2011, The genome of the mesopolyploid crop species Brassica rapa, Nature Genetics, 43(10): 1035-1039

https://doi.org/10.1038/ng.919
PMid:21873998

Xu W.J., Grain D., Bobet S., Le Gourrierec J., Thévenin J., Kelemen Z., Lepiniec L., and Dubos C., 2014, Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB-bHLH-WDR complexes and their targets in Arabidopsis seed, New Phytologist, 202(1): 132-144

https://doi.org/10.1111/nph.12620
PMid:24299194

Yan C.H., An G.H., Zhu T., Zhang W.Y., Zhang L., Peng L.Y., Chen J.J., and Kuang H.H., 2019, Independent activation of the BoMYB2 gene leading to purple traits in Brassica oleracea, Theor. Appl. Genet., 132(4): 895-906

https://doi.org/10.1007/s00122-018-3245-9
PMid:30467611

Yan M.L., Liu X.J., Guan C.Y., Chen X.B., and Liu Z.S., 2011, Cloning and expression analysis of an anthocyanidin synthase gene homolog from Brassica juncea, Molecular Breeding, 28(3): 313-322

https://doi.org/10.1007/s11032-010-9483-4

Yan M.L., Liu Z.S., Guan C.Y., Chen S.Y., Liu X.J., and Yuan M.Z., 2007, Cloning and sequence analysis of flavonoid biosynthesis genes in Brassica juncea, Zhongguo Nongye Kexue (Scientia Agricultura Sinica), 40(12): 2688-2695

Yang J.H., Liu D.Y., Wang X.W., Ji C.M., Cheng F., Liu B.N., Hu Z.Y., Chen S., Pental D., Ju Y.H., Yao P., Li X.M., Xie K., Zhang J.H., Wang J.L., Liu F., Ma W.W., Shopan J., Zheng H.K., Mackenzie S.A., and Zhang M.F., 2016, The genome sequence of allopolyploid Brassica juncea and analysis of differential homoeolog gene expression influencing selection, Nature Genetics, 48(10): 1225

https://doi.org/10.1038/ng.3657
PMid:27595476

Yang J.H., Zhang C.T., Zhao N., Zhang L.L., Hu Z.Y., Chen S., Zhang M.F., 2018, Chinese root-type mustard provides phylogenomic insights into the evolution of the multi-use diversified allopolyploid Brassica juncea, Molecular Plant, 11(3): 512-514

https://doi.org/10.1016/j.molp.2017.11.007
PMid:29183772

Yao G.F., Ming M.L., Allan A.C., Gu C., Li L.T., Wu X., Wang R.Z., Chang Y.J., Qi K.J., Zhang S.L., and Wu J., 2017, Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis, Plant J., 92(3): 437-451

https://doi.org/10.1111/tpj.13666
PMid:28845529

Zhang B., Hu Z.L., Zhang Y.J., Li Y.L., Zhou S., and Chen G.P., 2012, A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale (Brassica Oleracea var. acephala f. tricolor), Plant Cell Rep., 31(2): 281-289

https://doi.org/10.1007/s00299-011-1162-3
PMid:21987119