Mango (Mangifera indica L.) is known as the king of tropical fruits for its unique flavor. Its fruit has a variety of colors such as red, yellow, green, pink, yellow in red and green in yellow. Mango fruit color formation is related to metabolic pathways such as anthocyanin and carotenoids, of which the carotenoid synthesis pathway is involved in the color formation of fruits of various trees, including citrus (Peng, 2013; Zhu et al., 2017), persimmon (Zhao et al., 2011) and loquat (Hadjipieri et al., 2017). The analysis of different coloring mango varieties revealed that their fruits contained carotenoids, and it is speculated that carotenoid synthesis may be a key metabolic pathway for the coloring of mango fruits. Phytoene desaturase (PDS) catalyzes the dehydrogenation of Phytoene to generate 9,9'-dicis-ζ-carotene, followed by lycopene production via ζ-carotene desaturase (ZDS) and carotenoid isomerase (CRTISO), and PDS is involved in catalyzing the conversion of colorless Phytoene to colored carotenoids and the synthesis of other downstream carotenoids (Zhu et al., 2004). At the transcriptional level, the phytoenesynthase gene (psy), ζ-carotene dehydrogenase gene (zds) and phytoene desaturase (PDS) jointly regulate the accumulation of carotenoids in fruits (Kato et al., 2004).

Current studies have shown that PDS genes have been successfully isolated in many species, such as Narcissus tazetta (Chen et al., 2008, Molecular Plant Breeding, 6(3): 574-578), Carica papaya (Gao et al., 2009), Fragaria×ananassa (Zhu et al., 2011), Strelitzia reginae (Huang and Fan, 2013), Cucumis melo (Zhao, 2014), Brassica alboglabra (Sun et al., 2016), Anthurium andraeanum (Cui et al., 2016), Cephalotaxus hainanensis (Qiao et al., 2017), Fructus Gardeniae (Dong et al., 2018), Prunus persica (Liang et al., 2018), Acer palmatum (Lin et al., 2019), Abelmoschus esculentus (Li et al., 2019). However, the cloning of mango PDS gene has not been reported.

In this study, a PDS gene was cloned from mango fruit for the first time and its biological information was analyzed to further investigate the function of this gene and regulate carotenoid biomolecular synthesis in mango fruit.

1 Results and Analysis

1.1 Acquisition of PDS genes in mango

The full-length cDNA sequence of mango PDS gene was obtained by splicing the sequence information of 3' end and 5' end using RACE technique. Specific primers were designed to amplify the full-length cDNA sequence, and the electrophoretic strips were recovered and sequenced to produce a PDS gene cDNA sequence with length of 1 820 bp, which contains an open reading frame (ORF) with length of 1 650 bp and can encode 549 amino acids. The obtained gene sequences were compared with the NCBI-registered PDS protein sequences of pomelo (Citrus maxima (Burm) Merr.), tobacco (Nicotiana tabacum L.) and maize (Zea mays L.) (Figure 1), and the cloned gene was identified as PDS.

1.2 Bioinformatics analysis of mango PDS protein

The amino acid content of mango PDS proteins was analyzed using bioinformatic Bioedit software. The results showed that the alanine, leucine and valine were found to be high in the protein sequence (Figure 2). By predicting the secondary structure of mango PDS protein with the help of DNAMAN software, we found that most of the secondary structures of mango PDS protein were irregularly coiles and β-turn, and there were also a few α-helices (Figure 3). The hydrophilicity/hydrophobicity of the PDS protein was analyzed using the Kyte and Doolittle algorithms of Bioedit software, and the amino acid hydrophobicity (positive value) of the PDS protein was the highest at 1.76 and the hydrophilicity (negative value) was the lowest at -1.68, with the amino acid content mainly between the two values (Figure 4). The Conserved domains (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) of NCBI were used to find the conserved domain of the sequence. It was found that PDS protein contained binding domains such as PLN02612, COG3349, Amino_oxidase, and phytoene_desat (Figure 5). PDS proteins were predicted to be localized in the nucleus with the help of WoLFPSORT online software (http://www.genscript.com/wolf-psort.html). The possible conformations of the tertiary structure of the protein were predicted on the Swissmodel online software (http://swissmodel.expasy.org/) (Figure 6). Clustering analysis of the affinities by DNAMAN software showed that the PDS proteins of mango could be clustered into one group with closer affinity to the protein sequences of pomelo, muskmelon, and papaya (Figure 7). Its protein interactions were predicted according to STRING software and found to have interactions with PSY, ZDS, CRTISO and other proteins (Figure 8).

1.3 RT-PCR analysis of PDS gene in mango

RNA was extracted from the peels of different mango varieties, and the extracted RNA was used as a template for reverse transcription to cDNA, which was analyzed by RT-PCR. The results showed that the expression of PDS gene was higher in the red 'Guifei' mango peel, which was 1.51 times than the yellow 'Jinhuang' mango, and 1.88 times than the green 'Guiqi' mango, where the expression of PDS gene was less in the green 'Guiqi' mango peel (Figure 9). It is preliminarily speculated that the cloned PDS gene plays an important role in carotenoid synthesis in mango peel.

2 Discussion

Carotenoids are the main pigments in many flowers and fruits. Their species and content are important factors affecting fruit color and quality. Therefore, it is necessary to deeply study the key enzymes involved in carotenoid synthesis and analyze the regulation mechanism of carotenoid synthesis pathway. Studies have shown that phytoene desaturase (PDS) is an important branch in the carotenoid synthesis pathway and that inhibition of PDS expression leads to the accumulation of phytoene, which ultimately affects carotenoid synthesis. Therefore, PDS plays an important role in flower and fruit color development in plants (Bartley and Scolnik, 1995; Zhu et al., 2002; Kato et al., 2004). Disturbance of PDS affects the biosynthesis of chlorophyll, carotenoids and gibberellins, leading to albinism and dwarfism (Naim et al., 2018).

In this study, a phytoene desaturase (PDS) gene was successfully cloned from the fruit of mango. The full-length cDNA sequence of this gene is 1 820 bp, with an open reading frame of 1 650 bp, encoding 549 amino acids. Analysis of the cloned gene-interacting proteins using the STRING database revealed that PDS proteins interacted with PSY, ZDS, and CRTISO, which is consistent with the results of Kato et al. (2004). PDS amino acid sequences of different species were searched on NCBI, and by predicting the conserved structural domains, it was found that the selected sequences all contain a conserved binding domain at the C-terminus of the PDS protein to bind either the NAD/NADP or FAD conserved structural domains (Xi et al., 2013). Prediction of the conserved structural domain of the mango PDS protein revealed that it also contains the FAD conserved structural domain, suggesting that this conserved structural domain may be related to the function of PDS and that the PDS code is highly conserved during evolution. The main form of the PDS gene in the genome exists as a single copy, and its encoded protein is in a cystoid on the chloroplast (Grunewald et al., 2000). Verification of kiwi AcPDS1 protein localization in the cell membrane and cytoplasm using laser confocal experiments (Chen, 2019). In this study, the software predicted that the mango PDS protein is localized in the nucleus, which is different from the former PDS protein localization, indicating that as organisms evolve, the PDS protein is not only restricted to the chloroplast, but the structure becomes more complex, thus requiring analysis of different species to better understand the function of this gene.

In red papaya and yellow papaya ripe fruits, the PDS expression differed within the two varieties, with significantly higher expression in red-fleshed fruits than in yellow-fleshed fruits (Gao et al., 2009). RT-PCR analysis revealed that pds gene expression was highest in mature red fruits of strawberry, followed by flowers, and the lowest expression was in leaves. It is speculated that pds may play an important role in carotenoid accumulation in strawberry fruits and color development of flowers (Zhu et al., 2011). The carotenoids in the peel of papaya are mainly lutein and β Carotenoids, while the carotenoids in the pulp are mainly lycopene. PDS gene expression is up-regulated during papaya fruit ripening, and the orange-red color of papaya may be due to the accumulation of lycopene, β-carotene and β-cryptoxanthin (Shen et al., 2019). Using the cloned kiwifruit AcPDS1 gene, β-carotene content was significantly increased in transiently expressed tobacco leaves (Chen, 2019). In this study, PDS gene expression analysis was performed on three different mango varieties with large coloring differences, and it was found that the expression of PDS gene in different color mango peels had significant differences, with the highest expression in the red 'Guifei' variety, followed by yellow 'Jinhuang' variety, and the lowest expression in the green 'Guiqi' variety. This is the same as the former. PDS gene affects the accumulation of carotenoids, which plays a key role in the formation of red peel in mango. Further studies on the role of the PDS gene in mango fruit coloration require further validation of expression in transgenic plants.

3 Materials and Methods

3.1 Selection of materials and extraction of total RNA from mango

In this study, a total of 30 fully matured mango fruits (Mangifera indica L.) of ‘Guifei’, ‘Jinhuang’ and ‘Guiqi’ were selected from the Mango Resource Germplasm Nursery of the Ministry of Agriculture, Danzhou City, Hainan Province (20°02′45.97″ N, 110°11′38.39″ E). The mango peel was mixed and sampled with a razor blade, and the samples were immediately snap-frozen in liquid nitrogen and stored in a -80 °C refrigerator for backup. The RNA extraction kit of Tiangen was used to extract the total RNA from mango peel. In the extraction of RNA, the appropriate amount of RNase-free DNase I enzyme solution was added for the removal of residual DNA and dissolved in RNase-free water, and the integrity of RNA and whether DNA was completely removed were detected by 1.0% agarose gel electrophoresis.

3.2 Acquisition of full-length cDNA sequences of PDS genes in mango

The first strand cDNA of total mango RNA was synthesized by reverse transcription using the SMARTer™ RACE cDNA Amplification Kit (Cloontech) with the extracted total mango RNA as the template, and the cDNAs at the 3' and 5' ends were amplified, respectively. The PCR reaction program was set as follows: pre-denaturation at 94 °C for 4 min; denaturation at 95 °C for 50 s, denaturation at 50 °C for 50 s, extension at 72 °C for 2 min, 30 cycles, and final extension at 72 °C for 7 min. The reaction system was 25 µL, containing 10 PCR buffers (including Mg2+) 2.5 µL, 25 ng DNA template, 20 µmol primers, 1.0 U Taq DNA polymerase, and 5.0 mmol dNTPs. After electrophoretic analysis of the PCR products, the target gene fragments are recovered, ligated to the vector, transformed into the sensory state, identified and the positive clones are sequenced. Based on the sequence results obtained for the 3' and 5' ends of the gene, the full-length gene of the gene was spliced and specific primers were designed to increase the full-length gene sequence.

3.3 Bioinformatics analysis and prediction of PDS gene in mango

ORF finder on NCBI was used to search for open reading frame of PDS gene. The amino acid content of PDS proteins was analyzed using Bioedit software and the hydrophilic/hydrophobic analysis of amino acids was performed with the help of Kyte and Doolittle algorithms. DNAMAN software was used to model protein secondary structure and perform homologous sequence alignment and establish a phylogenetic tree. The tertiary structure model of PDS protein was predicted and constructed using SWISS-MODEL online software (http://swissmodel.expasy.org/). BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) on NCBI was used to search for homologous sequences of PDS proteins. Conserved domains of the PDS protein sequence were analyzed using Conserved domains (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) in NCBI. Localization of PDS proteins was predicted on WoLFPSORT online software (http://www.genscript.com/wolf-psort.html); their protein interactions were predicted using STRING software. STRING software was used to predict their protein interactions.

3.4 Expression analysis of PDS genes in mango

RNA from three mango varieties with different fruit colors was reverse transcribed into the first strand of cDNA under the action of reverse transcriptase, and primers were designed for RT-PCR amplification using primer5.0. Design the internal reference primer sequences used for RT-PCR analysis and the primers used for target gene amplification (Table 1). The PCR products were subjected to 1.0% agarose gel electrophoresis and detected using ethidium bromide staining, and then analyzed by gel imaging system and Quantity one software to determine the the relative expression.

Author's Contributions

YKL and ZZC are the experimental designers and executors of this study. YKL, ZZC, GAP, and LMF completed the data analysis and the first draft of the paper. LRX, HJF, and ZMY participated in experimental design and analysis of experimental results. ZZC is the architect and person in charge of the project, guiding experimental design, data analysis and paper writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the National Key Research and Development Program of China (2018YFD1000500), the National Natural Science Foundation of China (31471850), the Special Fund for Basic Research Operations of the Chinese Academy of Tropical Agricultural Sciences (1630032017005; 1630032017004) and the Protection of Tropical Crop Germplasm Resources of the Ministry of Agriculture (15RZZY-07).

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