Mango (Mangifera indica Linnaeus) is native to India, which is a large evergreen tree in Anacardiaceae. Mango fruit is a popular fruit in human life. Mango fruit is rich in sugar, protein, carotene, vitamin C and other nutrients. Terpenoids are the largest natural products of plants at present. Secondary terpenoids produced by isoprenoid biosynthesis pathway play an extremely important role in the growth and development of plants (Trapp and Croteau, 2001). The MEP pathway locates in the plastids of plant cells and provides precursors for the synthesis of terpenoids. While 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT) is a catalytic enzyme in the third step of the MEP metabolic pathway, and one of the key enzymes in the MEP pathway. MCT enzyme catalyzes Cytidine triphosphate and 2-C-methyl-D-erythritol 4-phosphate (MEP) forms 4-diphosphocytidyl-2-C-methyl-D-erythritol (Rohdich et al., 1999), which plays an important role in the MEP pathway.

The gene of MCT enzyme has been cloned in a variety of plants. MCT gene has been cloned in Lepidium apetalum (Zhao et al., 2016), Fritillaria cirrhosa (Zhang et al., 2018), Tripterygium wilfordii (Tong et al., 2015), Artemisia annua (Zhang et al., 2016) and Taxus chinensis (Jian et al., 2019). Song et al. (2018) transferred the RNAi vector of Tripterygium wilfordii MCT gene into Tripterygium wilfordii suspension cells, and verified that interference with the expression of MCT gene significantly inhibited the accumulation of triptolide and celastrol in Tripterygium wilfordii. Jian et al. (2019) used real-time quantitative PCR experiment to prove that the expression level of MCT gene in the green bark of Taxus chinensis was significantly higher than that in other tissues, and the expression level in leaves was significantly higher than that in stems and roots. Zhang et al. (2016) transformed Artemisia annua MCT gene into Arabidopsis thaliana and conducted overexpression experiments. The results showed that the contents of chlorophyll a, chlorophyll b and carotenoid were significantly increased in Arabidopsis thaliana, indicating that MCT gene plays an important role in the biosynthesis of terpenoids.

Considering that MCT gene exists widely in plants and plays an important role in terpenoid synthesis, it is inferred that MCT gene may also play an important role in mango. However, functional analysis of MCT gene in mango has not been reported. In order to identify MCT gene in mango and explore the evolutionary route and expression pattern of MCT gene, this study used BLAST and HMMER software to search 14 MCT genes from 12 plant genomes, and constructed species evolutionary tree and gene evolutionary tree to analyze the evolutionary route of MCT gene in plants. The MCT gene in mango was identified and its expression was analyzed to provide scientific basis for further study on the function of MCT gene in mango.

1 Results and Analysis

1.1 Protein domain identification and sequence alignment of mango MCT gene

A conserved domain PF01128.19 was identified in the amino acid sequence of Arabidopsis thaliana MCT gene (AT2G02500.1) by searching the Pfam database and named IspD in the Pfam database. In the total length of 302 amino acids, this domain was located in the 79-299 amino acid region, covering 221 amino acids, indicating that the latter half of Arabidopsis MCT gene was very conserved (Figure 1).

Figure 1 The site of PF01128.19 domain in AT2G02500.1 and Mi04g14720.1 gene

Two genes containing PF01128.19 domain were also identified in the mango genome: Mi11g18190.1 and Mi04g14720.1. The protein sequence of Mi11g18190.1 gene contained 142 amino acids, and its domain location was 69-128 with a domain length of 60 amino acids, indicating that the domain segment of Mi11g18190.1 gene was incomplete (Figure 2). The full length of the protein sequence of Mi04g14720.1 gene was 293 amino acids, and its domain location was 64-284 with a domain length of 221 amino acids, indicating that the protein domain of Mi04g14720.1 gene was complete and the latter half of the region was very conserved (Figure 1). Since a protein domain with incomplete structure could not produce physiological functions, we removed the incomplete Mi11g18190.1 gene sequence and named the mango Mi04g14720.1 gene with complete structure as MiMCT gene for subsequent analysis.

Figure 2 The alignment of Arabidopsis MCT protein and mango MCT protein sequences

1.2 Identification of MCT gene

In order to further identify the mango MCT gene, 11 species whose genome sequences have been published were searched, all of which had high assembly quality genomes and covered the evolution process from lower plants to higher plants (Figure 3). Plants included are as follows: Chlamydomonas reinhardtii is the representative of green algae, Physcomitrella patens is the representative of bryophyte, Selaginellae moellendorfii is the representative of pteridophyte, Ginkgo biloba is the representative of gymnosperms, Amborella trichopoda is the the representative of the basal species of angiosperms, Oryza sativa and Zea mays are the representative of monocots, Arabidopsis thaliana, Citrus sinensis, Malus domestica and Prunus persica are the representative of dicotyledons.

Figure 3 Phylogenetic tree of species used to identify MCT gene

1.3 Evolution of the MCT gene

In order to analyze the evolutionary relationship of MCT gene in plants, BLASTP was used to search the protein model files of each species genome, and the protein sequences containing PF01128.19 domain were retrieved. A total of 14 MCT gene protein sequences containing the complete domain were obtained. The phylogenetic tree of MCT gene was constructed by maximum likelihood estimate (MLE) (Figure 4). The number of MCT gene remained low in all species, and there was only one MCT gene in other species except for two protein sequences of MCT gene in Physcomitrella patens and Zea mays, indicating that MCT gene was strictly controlled during plant evolution. Moreover, green algae, bryophytes, pteridophyte, gymnosperms, dicotyledons, and monocotyledons clustered in one branch respectively, indicating that the evolutionary relationship of MCT gene was consistent with that of the species in which it was located. Moreover, the evolution of mango MCT gene Min_Mi04g147201.1 was closely related to the evolution of citrus MCT gene Csi_orange1.1g022657m.

Figure 4 Phylogenetic tree of MCT genes

1.4 Expression of MCT gene in Mango

Two mango varieties ‘guire-82’ and ‘hongyu’ were selected to compare the expression levels of MiMCT genes and to express heat maps from transcriptome data of mango MiMCT genes. The expression levels of flesh of ripening were very low in mango ‘hongyu’ and ‘guire-82’. In ‘guire-82’, the expression of peels of ripening, peels of developing, flesh of ripening was slightly higher than that of flesh of ripening. But on the whole, the expression level of MiMCT gene was lower in both peel and flesh of ‘guire-82’, while the expression level of MiMCT gene was the highest in flesh of developing of mango ‘hongyu’, followed by that in peel of developing. Therefore, the expression levels of developing peel and flesh were higher than that of ripening peel and flesh. The expression difference of MiMCT gene in ‘guire-82’ and ‘hongyu’ tissues was compared, and the results showed that the expression level of MiMCT gene in flesh of ripening of ‘guire-82’ was significantly lower than that in other tissues. However, the expression level of ‘hongyu’ was as follows: the flesh of developing was significantly higher than that of peel, the peel of developing was significantly higher than that of ripening, and the peel of ripening was significantly higher than that flesh of ripening (Figure 5).

Figure 5 Expression of MiMCT gene in peels and flesh of ‘hongyu’ fruits as well as ‘guire-82’

2 Discussion

2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT) is not only a key enzyme involved in the synthesis of isopentenyl pyrophosphate, but also a key enzyme in the MEP metabolic pathway of terpenoid biosynthesis (Rohdich et al., 1999). The results of protein domain analysis in this study showed that the conserved domain of mango MCT protein was consistent with that of other 11 species, including one PF01128.19 domain, belonging to glycosyltransferase (Liu and Mushegian, 2003), indicating that MCT gene was highly conserved. It is speculated that mngo MCT gene plays an important role in MEP pathway.

In order to systematically study the evolution of MCT genes in plants, this study searched MCT genes in 12 plant genomes from lower plants to higher plants based on the homology of MCT genes in different plants, and obtained 14 MCT genes in total. Among them, there was 1 MCT gene in each of 10 plant genomes, indicating that its copy number was strictly controlled during plant evolution.

From the phylogenetic tree of MCT gene, it can be seen that MCT gene exists in all stages of species from green algae to angiosperms, indicating that MCT gene evolves with plant evolution. And the MCT evolution process is consistent with the evolution of its species, which indicating that MCT genes evolves with the evolution of the species. And the number of MCT genes is very conservative, two MCT genes were only found in Physcomitrella patens in bryophytes and Zea mays, and only one MCT gene was found in the genomes of other species. This may be caused by the choice of adaptability of nature, speculated that MCT genes may have an integral role in the process of evolution.

According to the expression levels of MCT gene in two mango varieties ‘guire-82’ and ‘hongyu’, it can be seen that the expression levels of MCT gene in the developing stage are higher than those in the ripening stage, and the expression levels in the peel and flesh of ‘hongyu’ are higher than those in the peel and flesh of ‘guire-82’. Therefore, it can be inferred that the expression of MCT gene is different in different mango varieties and tissues, which may play a more important role in the development of mango and may also be related to chloroplast formation.

At present, there are no studies on the MCT gene in mango. In this study, representative species from lower plants to higher plants were selected to conduct identification and evolution research on the MCT gene in mango, and the MCT gene was preliminarily identified. The expression of MCT gene in two mango cultivars was analyzed to provide basis for further study on the function of mango MCT gene.

3 Materials and Methods

3.1 MCT protein sequence acquisition

In this study, genome data of Chlamydomonas reinhardtii, Physcomitrella patens, Selaginella moellendorfii, Amborella trichopoda, Oryza sativa, Zea mays, Musa acuminata, Arabidopsis thaliana, Citrus sinensis, Malus domestica and Prunus persica were downloaded from the Phytozome database (Goodstein et al., 2012). Ginkgo genome data download from http://gigadb.org/dataset/100209 (Guan et al., 2016). Mango genome was sequencing data in this study. BLAST was used to search the protein model database of each species, and the searched E value was set to 1e-10. Combined with HMMER software analysis, MCT gene protein sequence information of each species was obtained (Finn et al., 2011).

3.2 Identification and sequence modification of MCT protein conserved domain

The description line contents of protein sequences in each species were removed, only the sequence ID and sequence were retained, and the MCT gene protein sequences of each species were input into Pfam database. The default parameters were used as the standard to identify the domain and obtain the start and end site information (El-Gebali et al., 2018), and the sequences containing complete domain were retained. Sequence alignment was carried out to remove repeated sequences. The sequence ID of each species was labeled at the beginning as a combination of genus name and species name (Wang et al., 2013). For example, the MCT gene of mango is labeled Min_Mi04g147201.1, and the MCT gene of Arabidopsis is labeled Ath_AT2G02500.

3.3 MCT protein sequence alignment and phylogenetic tree construction

The determined protein sequences were compared by Probcons (Do et al., 2005), and the phylogenetic tree was constructed by PhyML. Bootstrap analysis of 1 000 generations was performed (Guindon et al., 2009). FigTree v1.4.4 was used to display the phylogenetic tree.

3.4 Expression analysis of MCT gene

In this study, mango varieties ‘guire-82’ and ‘hongyu’ were planted in the Mango Germplasm Resource Nursery of Danzhou, Hainan Province, and fresh peel and flesh samples of the two varieties at the development and maturity stages were collected respectively. The samples were three biological iterations, which were quick-frozen in liquid nitrogen and stored at -80℃ for reserve. Transcriptome sequencing data were extracted to compare the expression of mango MCT gene.

Authors’ contributions

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

Acknowledgments

This study was supported by the Hainan Provincial Natural Science Foundation of China (319QN310) and Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (1630032019033).

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