Genetic diversity of fluorescent protein genes generated by gene duplication and alternative splicing in reef-building corals
© Kariyazono-Takahashi et al. 2015
Received: 25 March 2015
Accepted: 17 June 2015
Published: 21 July 2015
Reef-building corals (Scleractinia) exhibit various colors, of which fluorescent proteins (FPs) are a major determinant. Gene duplication is considered a major mechanism in the generation of the FP gene family and color diversity. Examining gene duplication events and subsequent evolution may improve our understanding of FP gene family diversity.
We isolated a novel FP gene family from one individual of Montipora sp., which we named monGFP (GFP gene from Montipora sp.). This gene family consists of at least four genes that produce at least six different cDNA sequences. The sequences were categorized into two types based on the length of cDNA; this difference is attributed to alternative splicing. Although the amino acid sequences were different, the emission spectra of the monGFP variants were nearly identical (518–521 nm). In addition to this gene family, we isolated ten paralogous AdiFP10 (Adi-Fluorescent protein-10 gene from Acropora digitifera) sequences from cDNA of two Acropora species, A. digitifera and A. tenuis. Based on our phylogenetic analysis, five sequences from A. digitifera and four sequences from A. tenuis appeared to be in a different cluster from AdiFP10, suggesting a new FP gene cluster. The FP sequences were likely to have been generated independently in each species or generated by gene duplications in the ancestral lineage of Acropora, followed by extensive gene conversion within each species.
Our results clarify a part of the diversification process of FP genes during the evolutionary history of Montipora and Acropora species. Our analyses of monGFP indicate that FPs translated from different splicing variants and gene copies have evolved without changes in the function of fluorescence, and gene copies have been evolved under purifying selection. On the other hand, AdiFP10 paralogs and other RFP genes in Acropora species may have diversified their functions. Identification of conserved and divergent modes of evolution after the duplication of FP genes may reflect variation in the biological roles of different FPs.
In tropical shallow oceans, reef-building corals (Scleractinia) exhibit various colors. Fluorescent proteins (FPs) are a major color determinant in corals [1–3]; indeed, the pigments in different color morphs have been shown to be correlated with copy number variation in an FP gene . FPs are excited by environmental light and emit a longer wavelength of fluorescent light than the excitation light , and this fluorescent light can be perceived as “color”. FP color property is determined by amino acid sequences of FPs; in this sense, the evolution of FP genes represents the evolution of colors in corals .
The initial discovery and determination of the structure of FP was first studied in the crystal jelly, Aequorea victoria (synonym A. aequorea) [7, 8]. This protein, called green fluorescent protein (GFP), is excited by blue light (wavelengths shorter than 470 nm) and emits green fluorescence (maximum emission light: λemiss = 509 nm) . Since the discovery of FP in the crystal jelly, FP genes have been cloned from anthozoa , marine crustacean copepods , and deuterostome chordate amphioxus species [12–14]. The emission of florescence with longer wavelengths than excitation light is the most useful molecular tool for in vivo imaging techniques .
A variety of FPs has been detected in anthozoan species, with emission spectra in the visible light range. FPs are classified into four groups based on the color of light detected: cyan (CFP), green (GFP), yellow (YFP), and red (RFP) [16, 17]. Since blue/purple non-fluorescent chromoprotein is in the same phylogenetic cluster with other FP genes, it is also classified as an FP gene family member . The origin of FP genes pre-dates the divergence of coral families, and may have occurred as early as the Jurassic period . During coral evolution, diversification of fluorescent colors has occurred across species and FPs of the same fluorescence class have emerged repeatedly and independently; for example, CFPs, YFPs, and RFPs have evolved several times in different lineages .
In addition to fluorescent emission, many roles of FPs have been proposed; they are thought to be essential for viability, and may have photoprotective and antioxidant roles [4, 18–20]. However, detailed biological roles of FPs in corals remain unclear as the functional mechanisms have not been elucidated.
Gene duplication is a major source of genetic variation and can lead to neofunctionalization of the genes [21, 22]. Neo-functionalization is a mechanism under which one copy of a duplicated gene retains the original function, leaving the other copy free from purifying selection and able to acquire new functions . Beside the acquisition of a new function, gene duplication can also increase the transcription level by multiple copy number of genes, as is seen in the gene family that encodes ribosomal RNA . In FP genes, gene duplication is considered to be a major mechanism of generating FP gene family and color diversity . For example, ten FP genes have been isolated from the both genome and transcriptome of Acropora digitifera [24, 25]. Genomic analysis identified two regions, each of which consists of multiple copies of different FP genes . Different copies of amilFP597 genes are present in head-to-tail tandem arrangements in the Acropora millepora genome . The phylogenetic analysis of available FP gene sequences showed that FP gene duplication and the subsequent acquisition of various fluorescent colors has occurred multiple times, indicating repeated evolution of FP colors . In addition to the acquisition of different colors, copy number variation of amilFP597 is correlated with red pigment concentration , indicative of increase in FP of the same color. The study of gene duplications and subsequent genetic diversification process of FP genes is important for understanding FP gene family diversity.
Alternative splicing, in which mature mRNAs are generated from a single pre-mRNA transcript, is another mechanism that may generate variation of protein functions. Alternative splicing increases protein diversity because the mature mRNAs sometimes encode proteins with subtly different or opposing functional domains . However, alternative splicing of FP transcripts has not been reported, to our knowledge.
In this study, to evaluate the genetic diversification of the FP gene family, we analyzed gene duplication events and alternative splicing of FP genes in Scleractinia. We isolated novel GFP genes from Montipora sp., and identified a role of both gene duplication and alternative splicing in FP diversification. In addition to the GFP family, novel paralogs of an RFP gene were also identified in Acropora species. Our results shed new light on FP gene diversification during the evolutionary history of corals.
Materials and Methods
Specimens and species identification
Montipora sp. was acquired via an aquarium trade. The genus was predicted based on the sequence of the mitochondrial COI region (Additional file 1: Fig. S1) using the primers and method described in a previous study . The sequence of COI region was aligned by using Genetyx (ver. 16.0.0), and subjected to a phylogenetic analysis (neighbor-joining method ) with 1,000 bootstrap replications. Under permits from the Aquaculture Agency of Okinawa Prefecture (number 26–9), parts of an Acropora digitifera and A. tenuis colony were collected and subsequently maintained in an aquarium at the Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus. The two species were identified based on morphology.
DNA and RNA extraction
Genomic DNA was extracted from adult coral tissues of each species using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Total RNA was extracted from adult tissues and larvae of each species using the RNeasy Mini Kit (Qiagen).
Isolation of novel FP cDNA
cDNAs were synthesized using a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Shiga, Japan) from total RNA of Montipora sp.. An FP partial cDNA was cloned by RT-PCR using degenerate primers RFP_F2 (5’-ATGGAAGGGTSTGTCGATGG-3’) and RFP_R (5’-TKTTCTACCAGTYGCCATTTCTG-3’), and full-length cDNAs were isolated from the RNA of Montipora sp. by 5’ rapid amplification of cDNA ends (RACE) and 3’ RACE using the nested primers M5_RACE_F1 (5’-CAGTTCCACAGTACTTACAAGGCAAAGAC-3’), M5_RACE_R1 (5’-CATCAGGGCATGAGTTCTTGAAGTAGTC-3’), M5_RACE_F2 (5’-CGAAAGAGATGCCAGACTTCCACTTC-3’), and M5_RACE_R2 (5’-CAACTATGCCTCTGGGATATTTAGTGATG-3’), and a GeneRacer Kit (Invitrogen, Carlsbad, CA, USA). PCR was performed using the GeneAmp PCR System 9700 (Applied Biosystems, Carlsbad, CA, USA). All positions of primers are described in Fig. S2. The PCR reaction condition for partial cDNA was a denaturation step for 3 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C. The 5’ and 3’ RACE procedures were performed with two rounds of nested PCR using M5_RACE_F1 and M5_RACE_R1 for the first round, and M5_RACE_F2 and M5_RACE_R2 for the second round. The first and second PCRs were performed under the same condition, consisting of a denaturation step for 3 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 65 °C, and extension for 3 min at 72 °C. The novel FP cDNA was amplified using M5_F1 (5’-ATGCAAGCCAACAAATGCGCAA-3’) and M5_R1 (5’-CAAGGAGCTTGCAGATGCAGAAG-3’) primers and cDNA of Montipora sp. as a template. The PCR condition for the FP cDNA consisted of a denaturation for 3 min at 94 °C, followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C. The PCR product was purified for direct sequencing or cloned into the T-Vector pMD20 vector (Takara), and the sequences were determined using the Applied Biosystems Automated 3130 Sequencer.
Determination of the exon-intron structure of novel FP genes
The novel FP gene was amplified using M5_TC_LF (5’-GCAAACCGTGTGCTTGGATTCAC-3’) and M5_TC_LR (5’-CTTCCCACCAGTTGCCATTTCTG-3’) primers from the genomic DNA of Montipora sp. The PCR condition for the genomic FP gene sequence was denaturation for 3 min at 94 °C, followed by 30 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 65 °C, and extension for 10 min at 72 °C. The sequences of the whole intron 1, both ends of the other introns, and all exons were determined from the PCR product using M5eF1 (5’-CAACAAATGCGCAAAKAAGGCAAAC-3’), M5eR1 (5’- ACAGATCGCTTGATGCCTCAGAAG-3’), M5eR1_2 (5’- GGAATTTCAGGTCCATTTTGC-3’), M5eF2 (5’-TTGTATTACTTCTGAGGCATCAAG-3’), M5_RACE_R2 (5’-CAACTATGCCTCTGGGATATTTAGTGATG-3’), M5eF3 (5’-GGTTGACTATTTCAAGAACTCATG-3’), M5eR2 (5’- CATCACAGGTCCATTAGCAGGAAAG-3’), M5_TC_LR, and M5_GT_LR as sequencing primers. Partial intron 1 sequences between exon 1 and the additional 105 bp sequence (see results and discussion) were amplified using primers M5eF1 and M5eR1 from the genomic DNA of Montipora sp.. PCR products were cloned into T-Vector pMD20 and the sequences were determined using M5eF1, M5eR1, and M5i1_2_4F (5’-CGTCAAGATCCAGATTAACCTGT-3’) for all intron 1 sequences; M5i1L770 (5’-TGCTGAGACGGAAAATCTCAATGCT-3’) and M5i1-770R (5’-AGTCTGTTCTTAATAGCATTGAGA-3’) for i1_1250 (the name of intron 1 type differed by the length, see results and discussion); M5i1-1300R (5’-TAGATCTCAGGTTACGTAATTCTGA-3’) and M5i1_3R (5’-CGACAGCTGACGTGCGATCTAAC-3’) for i1_1800; M5i1_2R (5’-AGTTAAGACATACGACAATAACAAAACGCA-3’), M5i1L1700 (5’- TTAGCTCGTAGTGCCGTGGAAAGTTAG-3’), and M5i1-1700R (5’-AGTCACGCTCCATTAACTACGT-3’) for i1_2100; and M5i1_1R (5’-AAAGACATTATTAGATCTGTTACGTAATTT-3’), M5i1-2400 F (5’-TCTCAGTATAAAAGGTCCAAAGTC-3’), and M5i1-2400R (5’-TTAAAGAACTTAGTGTTCACTAG-3’) for il_2800. The rest of intron 1 sequences (downstream part) were amplified from the genomic DNA of Montipora sp., using the primer pairs of M5eR1_2 and specific to each intron type M5i1-2400 F, M5i1-1700 F, and M5i1-770. The rest of the intron 1 sequences were determined from the PCR product using M5i1_2_4F and M5eR1. The whole intron 1 sequences were amplified from the genomic DNA of Montipora sp., using a primer pair of M5eF1 and M5eR1_2. DNA fragments of each intron type from exon 1 through to intron 1, and intron 1 through to exon 3 were amplified from the genomic DNA of Montipora sp., using the primer pairs of M5F1 and specific to each intron type M5i1-2400R, M5i1-1700R, and M5i1-770R for exon 1, and those of M5_RACE_R2 and specific to each intron type M5i1-2400 F, M5i1L1700, and M5i1L770 for exons 2 and 3. The sequences of intron 2 and partial sequences of exon 1 and exon 3 were determined using the M5i1-2400R, M5i1-1700R, M5i1-770R, and M5i2LF (5′-CGAATAGCTCAAGTTGAACTCAAGCGA-3′) primers.
Determination of paralogs of AdiFP10 sequences from Acropora species
Complementary DNAs were synthesized using a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) from total RNA of A. digitifera and A. tenuis. A set of primers AdiFP10_F2 (5’-TACGGAAACAGGGTCTTCACTG-3’) and AdiFP10_R1 (5’-TGTTCTGCCAGTTGCCATTTCTG-3’) were designed based on AdiFP10 (accession number: BR000970). Using these primers and cDNA of A. digitifera and A. tenuis as templates, FP cDNAs were amplified. PCR products were cloned and the sequences were determined. The sequences were aligned using Genetyx (ver. 16.0.0). In order to remove PCR errors, cDNA sequences appeared at least two times were used for phylogenetic analysis.
Phylogenetic and evolutionary analysis
The coding sequence of the novel FP gene was aligned with FP genes from other Scleractinia species by using Genetyx (ver. 16.0.0) and ClustalW , and subjected to a phylogenetic analysis (maximum likelihood method ) with 1,000 bootstrap replications. Sites with gaps or missing data were eliminated from the whole sequences used in following analyses. dN/dS values were calculated by using MEGA 5 . The nucleotide sequences were deposited in GenBank under accession numbers LC029006 – LC029029.
Recombinant FP protein and spectroscopy
FP cDNAs were cloned into a pCold I expression vector (Takara), and then transformed into the BL 21 Escherichia coli strain (Takara). Each clone was grown in 30 ml of LB medium overnight, and the recombinant protein was extracted by sonication and centrifugation. The S2 sequence in the expression vector was constructed from the S3 sequence by in vitro mutagenesis. In vitro mutagenesis of FP was performed by extension of DNA synthesis of overlapped oligo-nucleotides using PCR . Emission spectra of the FP products were determined using a USB-4000 Spectrometer (Ocean Optics, Dunedin, FL, USA); the absorption spectra were measured using a Gene Spec V Spectrometer (Hitachi, Tokyo, Japan). Both fluorescence and absorption were measured three to six times for each protein except absorption of S2 (once). The absorption spectra were used as the approximate excitation spectra in this study.
Results and Discussion
Isolation of a novel FP gene
Alternative splicing and gene duplication of monGFP
To examine the functions of different genes and splice variants, we generated monGFP proteins from variants L1, L2, S1, and S2 to measure fluorescence. As shown in Fig. 4, the maximum excitation spectra (λexcit) and the maximum emission spectra (λemiss) of all monGFPs ranged from 505 nm to 507 nm, and from 518 nm to 521 nm, respectively. These results suggest that monGFPs translated from duplicated genes and alternatively spliced mRNAs exhibit nearly identical functions. The difference in λemiss between L1 and L2 monGFPs was 2 nm (Fig. 4a and b) and was attributed to a single amino acid replacement in exon 3 (Fig. 3c). The amino acid sequence from exon 2 to 5 of each variant pair of L1 and S1, or L2 and S2, was identical (Fig. 3c), but those of N terminal regions of S and L types were completely different (Fig. S3b). The fluorescence emitted from monGFPs of L1 and S1 was equivalent (Fig. 4a and c), and that of L2 and S2 was different by 2 nm (Fig. 4b and d). Although the length and amino acid sequences of the N terminal were different between S and L types, the differences in proteins attributed to alternative splicing do not cause a large difference in the wavelength of emitted fluorescence. FPs translated from different gene copies have been strongly conserved in the function of fluorescence. The analysis of the ratio of substitution rates at non-synonymous and synonymous sites (dN/dS) showed negative selection acting on this gene family (0.18 ± 0.01). These results suggest that monGFP genes have evolved under purifying selection.
The question of the biological role of gene duplication and alternative splicing of monGFP genes thus arises. One possible role is in increasing the expression of proteins with the same function. In A. millepora, the copy number variation of amilFP597 with a particular promoter type is correlated with red pigment concentration. The higher expression of amilFP597 has a photoprotective role to reduce photodamage to zooxanthellae under acute light . The other possibility is differential expression of splicing variants in different tissues. Although the fluorescent spectra were nearly identical, the increased gene copy number could increase the diversity in gene expression in different tissues  if each gene in the family is regulated independently. Since the quantity of the PCR products were nearly the same in the electrophoresis gel (Fig. 3a, S and L types), the expression levels of the two types of variants were roughly comparable, suggesting that both types may be functional in live corals. The two types of variants might have tissue-specific functions, similar to the muscle-specific roles of the splicing variants of the Mef2D gene .
Gene duplication of the AdiFP10 paralog
In this study, we showed that diversification of FP genes in Scleractinia occurred by gene duplication events and alternative splicing. We found at least four monGFP genes in the genome of Montipora sp., each with a similar function in terms of emitted fluorescence. Regarding the wavelength of fluorescence, monGFP genes have evolved without change in function even though the amino acid sequences of the first exon in different splicing variants are completely different. The similar function of duplicated monGFP may be attained through both purifying selection and homogenization by gene conversion, although the evidence supporting gene conversion in monGFP remains unclear. One of possible biological causes for the homogenization might be a requirement of increasing amount of the protein. Although the conserved evolution of the function of FPs is not well studied, this mode of evolution may be an important aspect with regard to FPs. When corals regulate the expression level of FPs to adapt or acclimatize to different environments, such as various light stresses , the functionally conserved multi-copy FP genes may be advantageous due to their ability to greatly change FP expression levels. On the other hand, gene duplication events and the acquisition of new functions has also been suggested in the FP family . AdiFP10 paralogs have diversified in Acropora species (Fig. 5). Although we did not measure the emission spectra of the newly identified paralogs, amilRFP and meffRFP possess distinct fluorescent spectra, with emission peaks at 593 nm and 576 nm, respectively , suggesting that AdiFP10 paralogs and other RFP genes in Acropora species may have diversified with respect to their functions, i.e., fluorescence. Thus, these FP genes are consistent with divergent evolution, as suggested previously . Our results regarding evolution of FP genes are consistent with various modes of evolution. These differences might reflect variation in the biological roles of different FPs. Further detailed evolutionary analysis of FP genes may reveal the roles of FPs in live corals.
This work was supported by an internal grant of SOKENDAI to Y.T. and an IRC grant of SOKENDAI to S. T. K. We thank Drs. Tatsuya Ota (SOKENDAI) and Hideyuki Tanabe (SOKENDAI) for help with the experiments, and Masayuki Hatta (Ochanomizu University) for help with sampling.
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