Cloning and Expression Analysis of PAP1 in Brassica juncea

Anthocyanins are important substances accounting for the leaf color in Brassica juncea and PAP1 gene is one of the key transport factors in the anthocyanin synthesis pathway. In this study, homologous cloning technology was used to clone the PAP1 gene sequences of Brassica juncea with different leaf colors. Specific primers were designed according to the gene sequences of Brassica rapa with high homology for PCR amplification. The PAP1 gene of Brassica juncea is 1 348 bp~1 669 bp long, and the coding region sequence is 744 bp~753 bp, including 3 exons and 2 introns. Two MYB blinding domains are found in PAP1 protein at the site of 9~59 and 62~110 amino acids. Phylogenetic analysis showed that the PAP1 gene of Brassica juncea had high homology with the related genes of Brassica rapa and Brassica rapa subsp. rapa, but had low homology with Arabidopsis thaliana. Compared gene sequences in Brassica juncea with different leaf colors, there are no differences between the coding sequence of purple and red leaf Brassica juncea, but the encoded protein have 22 amino acid differences from green leaves. We also observed the lower expression level of PAP1 and its related target genes such as DFR, TT19 in green leaves, which may lead to the differences of leaf color in Brassica juncea. This study provides a reference for exploring the function of PAP1 gene and the formation mechanism of different leaf color of Brassica juncea.

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 1 Results and Analysis

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.

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.

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).

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). 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.   (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)

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. 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.

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 . 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.

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.

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.

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.

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.