The major royal jelly protein (MRJP) is a group of proteins for royal jelly (Wang et al., 2020); these proteins are produced only by honey bees(Corzo et al., 2023). MRJP proteins are encoded by nine related genes: mrjp1-mrjp9 (Helbing et al., 2017). The mrjp family genes are grouped tandemly in a total of 60 kb base pairs and evolved from a single progenitor, i,e. the yellow protein gene family via gene duplication (Drapeau et al., 2006). This gene evolution conserves the exon/intron structure of the mrjp gene family (Drapeau et al., 2006). Although mrjp genes have similar exon/ intron structures, each gene produces a protein with unique characteristics and functions (Botezan et al., 2023). The molecular weights of MRJP1, MRJP2, MRJP3, and MRJP5 proteins are 53 kDa, 46.5 kDa, 66.8 kDa, and 80.9 kDa, respectively (A et al., 2022).

Mrjp genes encode proteins that are responsible for honeybee nutrition, physiological function, and the development of social behaviors such as caste determination (Buttstedt et al., 2013), as well as the division of labor for nursing and foraging among honeybee workers (Fang et al., 2023). The proteins of MRJP2 (Feng et al., 2021), MRJP8, and MRJP9 (Lee et al., 2022) show antimicrobial activities that are important for honey bee larval immune systems. Besides antimicrobial activity, the recombinants of Apis mellifera Linnaeus MRJP’s 1-7 provide antioxidant activity against H₂O₂(Park et al., 2020). Additionally, MRJP proteins have several health benefits, e.g, anti-tumor(Abu-Serie & Habashy, 2019), anti-aging (Jiang et al., 2018), anti-inflammatory (Hayashi et al., 2011), and antiapoptotic (Kim, 2021); summarized by (Mureşan et al., 2022).

The most common gene in the mrjp family is mrjp2 which encodes major royal jelly protein 2 (MRJP2) (Schmitzova et al., 1998). Mrjp2 gene sequences of Apis cerana Fabricius and A. mellifera show high polymorphism (Su et al., 2005). This gene was used to detect the entomological origin of A. cerana honey from China and A. mellifera honey from China, Brazil, Australia, and South Africa (Zhang et al., 2019) and also successfully distinguish the honey from those both bees in Java, Indonesia (Raffiudin et al., 2023). The first mrjp2 gene DNA sequences of A. cerana and A. mellifera originated from Indonesia and have been published in the GenBank (NCBI) database (Raffiudin et al., 2023). Another study found that there are three specific peptide markers of mrjp1, mrjp2, and mrjp3 genes for honey authentication, namely YNGVPSSLNVISK, TLQMIAGMK, and LTVAGESFTVK, respectively (Jiang et al., 2021).

Currently, the mrjp2 gene database at NCBI is limited to mRNA and Coding (CDS) sequences, with little information regarding exon/intron structure. Therefore, this study aims to characterize and analyze the exon/intron organization, nucleotide variation, variations in putative amino acids, and the phylogeny of A. cerana and A. mellifera bees in Indonesia based on their mrjp2 genes. Relationships with other mrjp family genes is also examined.

Both mrjp2 gene sequences of A. cerana and A. mellifera bees have higher AT nucleotide content than GT nucleotide content, consistent with (Raffiudin et al., 2022). The whole genome A. mellifera also reported that honey bees contain high amounts of adenine (A) and thymine (T) compared to other insect genome sequences (Honeybee Genome Sequencing Consortium 2006). The results of the BLAST-N DNA sequences of A. cerana and A. mellifera showed that both were homologous to the samples of A. cerana (LC596999.1) and A. mellifera (LC600169.1) from Java, Indonesia with 100% homology values and an E-value of 0. A nucleotide sequence can be said to be homologous if the results of homology analysis using BLAST-N show an E-value close to zero (Pearson, 2013)or an identity value greater than 70% for nucleotide sequence data and more than 25% for the analyzed amino acid sequence data (Claviere & Notredame, 2007). The high homology of A. cerana is consistent with the previous results that found there is no variation of the mrjp2 gene in A. cerana from several provinces in Indonesia (Raffiudin et al., 2022)

Alignment results showed that A. cerana and A. mellifera sequences comprised the exon 1 to partial exon 3, this result confirmed the targeted mrjp2 region of MF-MR primer (Zhang et al., 2019). The intron region of A. cerana and A. mellifera mrjp2 gene sequences have fulfilled the GT-AG consensus (Kitamura-Abe et al., 2004), that flank the exon region (Breathnach et al., 1978). The resulting of 112 and 123 putative amino acids in exon 2 and 3, respectively of both honey bee were confirmed by using InterPro as the MRJP family member. The MRJP protein contains around 400–578 putative amino acids (Buttstedt et al., 2013) with the mrjp2 gene having six exons and five introns (Drapeau et al., 2006). This study found that the first 14 putative amino acids were detected as the N-terminal signal peptide both in A. cerana and A. mellifera. This signal is supported by the study that revealed the MRJP family consists of 16–20 amino acids N-terminal signal peptide (Schmitzova et al., 1998); (Buttstedt et al., 2014). A recent study found that there were three N-glycosylation sites in MRJP2 isolated from fresh royal jelly of A. mellifera, which are N145, N178, and N₉₂ (Lin et al., 2019). Based on (Lin et al., 2019), our result confirmed that the MRJP2 putative amino acid of A. cerana and A. mellifera showed the expected N-glycosylation site in the asparagine (N) amino acid number 92. The N-glycosylation has been conserved and is important since these asparagine residues are the attachment sites for complex sugars or glycosylation (Park & Zhang, 2011).

Variations between A. cerana and A. mellifera in their nucleotide sequences and putative amino acids may reflect differences in the quantity and composition of the MRJP proteins that make up royal jelly (Yu et al., 2009). Moreover, the results show that A. cerana has more within-species nucleotide and amino acid variation than A. mellifera. This high genetic variation in A. cerana nucleotide sequences might be due to a wider geographical distribution and the presence of different subspecies. Variation within subspecies of A. cerana in Indonesia is low (Raffiudin et al., 2022).

The genetic distance results show that A. cerana bee samples have a genetic distance that was closer to the DNA sequences of the A. cerana mrjp2 gene than to the DNA sequences of the A. mellifera mrjp2 gene (Supplementary 1). This result follows (Su et al., 2005) that the mrjp2 gene of A. cerana and A. mellifera has a large polymorphism.

Although the mrjp2 gene of A. cerana and A. mellifera have a high polymorphism, however, they formed the monophyletic clade. Uniquely, the phylogenetic topology of A. cerana based on exons 2 and 3 of the mrjp2 gene showed that A. cerana from Bogor clustered with A. cerana (Gen ID 107997173) with a bootstrap value of 77%. This clade was separate from the other mrjp2 genes of A. cerana and A. melliferaFigure 4. Furthermore, the topology of the mrjp family gen tree showed that the A. cerana and A. mellifera mrjp2 clustered with the A. mellifera mrjp7 gene with a low bootstrap value of 41%Figure 4. The entire mrjp2 and mrjp7 family genes in the phylogenetic tree can still be classified as one cluster because the resulting bootstrap value is relatively lowFigure 4. Clusters in a phylogenetic tree are said to be real and reliable if they have a bootstrap proportion value of more than 70% (Hillis & Bull, 1993). The clustering of mrjp2 and mrjp7 genes are congruent with the phylogenetic tree of Apis spp. using the mrjp family that showed mrjp2 is grouped in the same cluster as mrjp7 (Drapeau et al., 2006); (Buttstedt et al., 2014).

The mrjp family gene tree also revealed that mrjp3+mrjp6 and mrjp5 genes were closely related. The mrjp3+mrjp6 group is similar to (Buttstedt et al., 2014) study, while the position of mrjp5 is inconsistent (Drapeau et al., 2006). On the other hand, mrjp8 and mrjp9 were clustered forming a group that is separated from other mrjp family membersFigure 4. This is consistent with previous studies (Drapeau et al., 2006); (Buttstedt et al., 2014). Perhaps the functions of mrjp8 and mrjp 9 differ from mrjp1-7. Unlike other mrjp family genes, the expression of mrjp8 and mrjp9 were low both in the brain and hypopharyngeal gland, while expressed in another organ, leading to the hypothesis that the function of those genes differs from other mrjp genes(Dobritzsch et al., 2019). The gene sequence of the mrjp families A. cerana and A. mellifera can group in the same cluster because the mrjp family genes evolved from one gene of the same family. The mrjp gene family of Apis spp. evolved from a common background, thus they have a close relationship (Drapeau et al., 2006). The mrjp gene family forms a large cluster with the mrjpl/yellow gene family in the Hymenopteran species, which includes Apis honey bees, non-Apis bees, and ants (Buttstedt et al., 2014). Knowledge of the mrjp gene family’s structure and evolution is important for our understanding of their roles in honey bee development and nutrition.

This study successfully isolated 579 bp and 597 bp of mrjp2 genes from A. cerana and A. mellifera, respectively. Those sequences are homologs with the mrjp2 gene of A. cerana and A. mellifera from Indonesia. They are comprised of partial exons 1 up to partial exon 3, and produce 111 and 123 putative amino acids of A. cerana and A. mellifera, respectively. Nucleotide and putative amino acid variation in A. cerana is higher than in A. mellifera. Phylogenetic tree construction showed that the A. cerana and A. mellifera mrjp2 genes have a close phylogenic relationship with the A. mellifera mrjp7 gene. The structure and phylogenetic relationships confirm that this family of genes has conserved exon/intron structure through gene duplication.