Terpenoids are the most abundant natural products of plants and play an important role in the growth and development of plants (Trapp and Croteau, 2001). Terpenoids are synthesized via the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol-4-phosphate (MEP) (Laule et al., 2003). 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) is an important key enzyme in the MEP pathway, catalyzing the conversion of 4-(cytidine 5-pyrophosphate)-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP) to 2-C-methylerythritol 2,4-cyclopyrophosphate (ME-CPP) (Gao et al., 2012).

MDS genes have been cloned in a variety of plants including Salvia miltiorrhiza (Gao et al., 2012), Eucommia ulmoides (Wang et al., 2017), Artemisia annua (Wang et al., 2013), and Ginkgo biloba (Kim et al., 2006), and their functions have been investigated in a variety of plants. Gao et al. (2012) increased tanshinones after inducing the hairy roots of Salvia miltiorrhiza with Ag⁺. Zhang (2016) transformed MDS gene from Artemisia annua into Arabidopsis thaliana. And the over expression experiment results showed that the content of chlorophyll a, chlorophyll b and carotenoids in Arabidopsis thaliana was increased, which proved that MDS gene plays an important role in the biosynthesis of terpenoids. In Catharanthus roseus, up regulation of MDS gene can increase the content of monoterpene indole choline in suspension-cultured cells, indicating that MDS gene is related to the biosynthesis of monoterpenoids (Veau et al., 2000). Liu (2008) used semi-quantitative RT-PCR experiments to show that the MDS gene was expressed in the bark, stems, leaves and roots of Taxus×media Rehder, but the expression was highest in the leaves and lowest in the stems, and the function of the taxol MDS gene in promoting β-carotenoid production and accumulation was verified in E. coli strains by prokaryotic expression experiments. Peng et al. (2008) also demonstrated that the ginkgo MDS gene can produce β-carotene by functional complementation experiments in E. coli.

At present, the function of MDS family genes in mango (Mangifera indica) has not been reported yet. In order to identify the MDS family genes in mango and study the evolution process and expression patterns of MDS genes, this study used the method of homologous search to identify 16 MDS family genes from 13 plant genomes and constructed a phylogenetic tree for MDS family genes, and carried out the analysis of the expression of MDS family genes in mango varieties 'Guire-82' and 'Hongyu', providing scientific basis for the subsequent study of the functions of mango MDS family genes.

1 Results and Analysis

1.1 Identification and sequence alignment of mango MDS family genes

The amino acid sequence of the known Arabidopsis MDS gene (AT1G63970.1) was downloaded and its protein structural domain was identified in the Pfam database. The results showed that the amino acid sequence of the Arabidopsis MDS gene contains a structural domain numbered YgbB domain with the number of PF02542.16. YgbB protein domain has the same N-terminal characteristics in eukaryotes and prokaryotes. Its main function is to participate in the biosynthesis of terpenoids and isoprenoids, and it is an important chemical substance in all organisms (Herz et al., 2000).

With the amino acid sequence of Arabidopsis MDS gene as the reference sequence, two genes containing PF02542.16 domain were identified in mango genome through BLAST mango genome protein data and HMMER software, namely Mi20g01650.1 andMi03g02020.1. Mi20g01650.1 contains 699 nucleotides and encodes 232 amino acids, and the YgbB protein domain is located in the 76-229 amino acid region. Mi03g02020.1 contains 717 nucleotides and encodes 238 amino acids, and the YgbB protein domain is located in the 82-235 amino acid region. The length of the domain is 154 amino acids, and it has a complete YgbB protein domain (Figure 1). Therefore, Mi20g01650.1 and Mi03g02020.1 genes were identified as mango MDS family genes. By comparing the nucleotide sequences of Mi20g01650.1 and Mi03g02020.1 genes, we found that their sequence consistency was 86.53% (Figure 2), including 3 exons and 2 introns (Figure 3).

1.2 Evolution of MDS gene

To analyze the evolutionary route of MDS family genes in plants, we also searched 12 plant genome databases through BLAST software, including Chlamydomonas reinhardtii, Physcomitrella patens, Selaginellae moellendorfii, Ginkgo biloba, Amborella trichopoda, Oryza sativa, Zea mays, Musa acuminata, Arabidopsis thaliana, Citrus sinensis, Malus domestica, and Prunus persica, which covered plants in the evolutionary process from Chlorophyta to Angiospermae, and combined with mango MDS family genes to obtain 16 MDS family genes. Phylogenetic tree was constructed with PhyML (Figure 4).

It can be seen from the phylogenetic tree that Chlamydomonas reinhardtii, the representative plant of Chlorophyta, has become an independent branch. Ginkgo biloba (the representative of Gymnosperms), Physcomitrella patens (the representative of Bryophyte), and Selaginellae moellendorfii (the representative of Pteridophyta) gather in one branch. Monocots and Dicots gather in one branch respectively, and two genes of mango MDS family gather together, and the closest one to mango is citrus. It can be inferred that the evolution of MDS gene is different from that of plant species and does not completely follow the evolutionary path from lower plants to higher plants. However, the MDS family genes in mango gradually evolved after the formation of dicots and are most closely related to the MDS genes in citrus.

1.3 Expression of MDS family genes in mango

The analysis of MDS family gene expression was conducted in mango varieties 'Guifei', 'Tainong' and 'Jinhuang'. The qPCR experimental data of two members of mango MDS family genes Mi03g02020.1 and Mi20g01650.1 in the flesh of ripe fruits of three varieties were mapped (Figure 5). The results showed that the two genes had the highest expression in 'Guifei', followed by 'Tainong', and the lowest expression in 'Jinhuang', and the expression of each gene was significantly different in the three varieties.

2 Discussion

Isoprenoid is an essential compound in all organisms. MDS is an enzyme in the isoprenoid synthesis pathway (Kishida et al., 2003). However, it has not been reported in mango. In this study, the analysis of MDS protein domain showed that the protein domain of mango MDS family genes are the same as that of other 12 plants, including one YgbB domain. The main function of YgbB domain is to participate in the biosynthesis of terpenoids (Herz et al., 2000). Therefore, it is speculated that mango MDS family genes play an important role in terpenoid synthesis pathway.

Due to the homology between MDS family genes of different species, this study used the method of homologous search to download 16 MDS family genes from the protein files of 13 plant genomes. These plants contain representative plants in the evolutionary process from Chlorophyta to Angiospermae. Except for Zea mays, Musa acuminata and Mangifera indica with 2 MDS genes, there are only one MDS gene in all plants, indicating that MDS family genes are extremely conservative in the evolutionary process, and the number of copies is strictly controlled.

The results of the phylogenetic tree of MDS family genes showed that Chlorophyta become an independent branch, Gymnosperms, Bryophyte and Pteridophyta gather in a branch, and monocots and dicots gather in a branch, respectively. The overall structure of the phylogenetic tree does not follow the evolutionary process from lower plants to higher plants, indicating that the evolution of MDS family genes is different from that of their species. Moreover, the two genes of mango MDS family are clustered together, and they are in a branch with other dicots, and have the closest relationship with citrus. Since mango and citrus grow in tropical and subtropical regions, it is speculated that the evolution of MDS family genes may be related to geographical climate.

The analysis results of mango MDS family gene expression in mango varieties 'Guifei', 'Tainong' and 'Jinhuang' showed that two members of mango MDS family genes have the highest expression amount in the flesh of 'Guifei' rape fruit, followed by the 'Tainong', and the lowest expression amount in 'Jinhuang', and the expression amount of each gene in the three varieties is significantly different, indicating that the that the expression of the same gene is different among different varieties, which may affect the differences among varieties.

In this study, the MDS family genes of mango were identified, and the evolutionary process of the MDS family genes in plants and the expression of the MDS family genes of mango were analyzed, providing a scientific basis for the subsequent study of the functions of the mango MDS family genes.

3 Materials and Methods

3.1 Download of MDS protein sequence

In this study, the protein data of Chlamydomonas reinhardtii, Physcomitrella patens, Selaginellae moellendorfii, Amborella trichopoda, Oryza sativa, Zea mays, Musa acuminata, Arabidopsis thaliana, Citrus sinensis, Malus domestica and Prunus persica were downloaded from Phytozome database. Ginkgo biloba genome data was downloaded from the website (http://gigadb.org/dataset/100209) (Guan et al., 2016). Mango genome is the data obtained from genome sequencing of this research group (https://www.ncbi.nlm.nih.gov/genome/?term=Mangifera+indica).

3.2 Identification and sequence ID of MDS protein domain

Using the method of homologous search, through BLAST various plant protein model databases, with the E value 1e-10. Matched the MDS protein sequence of each species with Pfam-A database, identified the protein domain of the MDS family gene (El Gebali et al., 2018), retained the sequence with the complete protein domain, removed the duplicate sequence, and obtained a total of 16 MDS family gene sequences. The protein sequence of each species was named according to the combination of genus name and species name (Wang et al., 2013). Mango MDS family gene markers are Min_Mi03g02020.1 and Min_Mi20g01650.1.

3.3 Construction of phylogenetic tree of MDS genes 

The MDS protein sequences of various species were compared (Do et al., 2005), and the sequence format was converted to the phylip. The MLE (Maximum Likelihood Estimate) was used to construct the phylogenetic tree (Guindon et al., 2009), and FigTree v1.4.4 was used to label the phylogenetic tree.

3.4 Expression analysis of MDS family genes in mango

In this study, mango varieties 'Guifei', 'Tainong' and 'Jinhuang' were planted in Hainan Danzhou Mango Germplasm Resource Nursery. Fresh samples of rape fruit flesh of each variety were collected respectively. The samples were all three biological replicates. After quick freezing in liquid nitrogen, they were stored in -80 °C refrigerator for use. The expression of MDS family genes in three mango varieties was compared by real-time fluorescent quantitative PCR. Primer sequence of MinActin internal reference gene was as follows: MinActin-F: 5’-ACCACCACTGCTGAACGGG-3’, MinActin-R: 5’-CCGATGAGTGATGGCTGGAA-3’; Mi20g01650.1-F: 5’-TTAGTATTCCCCACGACAGAGGTT-3’, Mi20g01650.1-R: 5’-AGTCCCAATGCTCCCAAGATAG-3’; Mi03g02020.1-F: 5’-TCACTGCCTTTTCGTGTGG-3’, Mi03g02020.1-R: 5’-GCCTCGCAACCTCTATCGT-3’.

The reaction procedure was set as follows: 95 °C for 30 s; 95 °C for 5 s and 60 °C for 30 s, with a total of 40 cycles. The reaction system was 25 μL in total, including TB Green Premix Ex Taq II (2×) 12.5 μL, upstream and downstream primers (10 μM) 1 μL each, cDNA 2 μL, dH₂O 8.5 μL. 2^-ΔΔCt was used to calculate the relative expression of genes.

Authors’ contributions

GJT is the experimental designer and executor of this study. GJT completed the data analysis and the writing of the first draft of the paper. CYY, WP, YXX, and LRX participated in the experimental design and the analysis of the experimental results. GJT is the designer and director of the project, guiding experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the Youth Fund Program of Hainan Natural Science Foundation (319QN310) and Special Fund for Basic Scientific Research of Chinese Academy of Tropical Agricultural Sciences (1630032019033).

References

Do C.B., Mahabhashyam M.S., Brudno M., and Batzoglou S., 2005, ProbCons: probabilistic consistency-based multiple sequence alignment, Genome Res., 15(2): 330-340.

https://doi.org/10.1101/gr.2821705

PMid:15687296 PMCid:PMC546535

El-Gebali S., Mistry J., Bateman A., Eddy S.R., Luciani A., Potter S.C., Qureshi M., Richardson L.J., Salazar G.A., Smart A., Sonnhammer E.L.L., Hirsh L., Paladin L., Piovesan D., Tosatto S.C.E., and Finn R.D., 2018, The Pfam protein families database in 2019, Nucleic Acids Res., 47(D): 427-432. 

https://doi.org/10.1093/nar/gky995

PMid:30357350 PMCid:PMC6324024

Gao W., Cheng Q.Q., Ma X.H., He Y.F., Jiang C., Yuan Y., and Huang L.Q., 2012, Cloning and bioinformatics analysis of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene in Salvia miltiorrhiza, Zhongguo Zhongyao Zazhi (China Journal of Chinese Materia Medica), 37(22): 3365-3370. 

Guan R., Zhao Y., Zhang H., Fan G., Liu X., Zhou W., Shi C., Wang J., Liu W., Liang X., Fu Y., Ma K., Zhao L., Zhang F., Lu Z., Lee S.M.Y., Xu X., Wang J., Yang H., Fu C., Ge S., and Chen W., 2016, Draft genome of the living fossil Ginkgo biloba, Gigascience, 5(1): 5-49.

https://doi.org/10.1186/s13742-016-0154-1

PMid:27871309 PMCid:PMC5118899

Guindon S., Delsuc F., Dufayard J.F., and Gascuel O., 2009, Estimating maximum likelihood phylogenies with PhyML, Bioinformatics for DNA Sequence Analysis, 537: 113-137.

https://doi.org/10.1007/978-1-59745-251-9_6

PMid:19378142

Herz S., Wungsintaweekul J., Schuhr C.A., Hecht S., Lüttgen H., Sagner S., Fellermeier M., Eisenreich W., Zenk M.H., Bacher A., and Rohdich F., 2000, Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate to 2C-methyl-D-erythritol 2, 4-cyclodiphosphate. Proc. Natl. Acad. Sci. USA, 97(6): 2486-2490.

https://doi.org/10.1073/pnas.040554697

PMid:10694574 PMCid:PMC15955

Kim S.M., Kuzuyama T., Chang Y.J., and Kim S.U., 2006, Cloning and characterization of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECS) gene from Ginkgo biloba, Plant cell Reports, 25(8): 829-835. 

https://doi.org/10.1007/s00299-006-0136-3

PMid:16528563

Kishida H., Wada T., Unzai S., Kuzuyama T., Takag, M., Terada T., Shirouzu M., Yokoyama S., Tame J.R.H., and Park S.Y., 2003, Structure and catalytic mechanism of 2-C-methyl-D-erythritol 2, 4-cyclodiphosphate (MECDP) synthase, an enzyme in the non-mevalonate pathway of isoprenoid synthesis, Acta Crystallogr. D. Biol. Crystallogr., 59(1): 23-31.

https://doi.org/10.1107/S0907444902017705

PMid:12499535

Laule O., Fürholz A., Chang H.S., Zhu T., Wang X., Heifetz P.B., Gruissem W., and Lange B.M., 2003, Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana, Proc. Natl. Acad. Sci. USA, 100(11): 6866-6871.

https://doi.org/10.1073/pnas.1031755100

PMid:12748386 PMCid:PMC164538

Liu W.H., 2008, Cloning and characterization of two key enzyme genes involved in the biosynthesis pathway of taxol precuresors, Thesis for M.S., Southwest University, Supervisor: Liao Z.H., and Chen M., pp.50-51.

Peng M.F., Yang Y.J., Yang C.X., Chen M., and Liao Z.H., 2008, Cloning and functional analysis of a new IspF gene from Ginkgo biloba, Linye Kexue (Science Silvae Sinicae), 44(10): 49-54. 

Trapp S.C., and Croteau R.B., 2001, Genomic organization of plant terpene synthases and molecular evolutionary implications, Genetics, 158(2): 811-832. 

https://doi.org/10.1093/genetics/158.2.811

PMid:11404343 PMCid:PMC1461696

Veau B., Courtois M., Oudin A., Chénieux J.C., Rideau M., and Clastre M., 2000, Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in catharanthus roseus, Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1517(1): 159-163.

https://doi.org/10.1016/S0167-4781(00)00240-2

Wang L., Du H.Y., and Wuyun T.N., 2017, Subcellular localization and expression analysis of genes from eucommia ulmoides involved in MVA and MEP pathway, Zhiwu Yanjiu (Bulletin of Botanical Research), 37(1): 52-62. 

Wang P., Wang Z., Dou Y.C., Zhang X.X., Wang M.Y., and Tian X.M., 2013, Genome-wide identification and analysis of membrane-bound O-acyltransferase (MBOAT) gene family in plants, Planta, 238(5): 907-922.

https://doi.org/10.1007/s00425-013-1939-4

PMid:23928653

Wang Y., Jia W.Z., Tan M.J., Zhang J., Wang D., and Liu S.H., 2013, Cloning and sequence analysis of MCS gene in Artemisia annua and its prokaryotic expression, Zhongcaoyao (Chinese Traditional and Herbal Drugs), 43(16): 2288-2293. 

Zhang M., 2016, Molecular cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene, 4-(cytidine 5'-diphospho)-2-C-methylery-thritol kinase gene, 2-C-methylerythritol-2，4-cyclodiphosphate synthase gene from Artemisia annua L, Thesis for M.S., Southwest University, Supervisor:Liao Z.H., pp.62-64.