بررسی چندشکلی ژن MRJP1 مرتبط با صفت تولید ژل رویال در زنبور عسل ایرانی (Apis Mellifera Meda) و تحلیل ارتباط آن با تولید ژل رویال

Introduction
Honey bees are indispensable insects that provide essential pollination services for agricultural crops while maintaining the balance of natural ecosystems. In this ecological role, they produce valuable products, including honey, beeswax, propolis, and, particularly, royal jelly, which holds exceptional economic and nutritional significance. Royal jelly is a gelatinous, yellowish-white substance secreted from the hypopharyngeal and mandibular glands of nurse worker bees. This secretion serves as the exclusive food source for the queen honey bee throughout her lifespan. It constitutes the primary nourishment for all larvae during their first three days of development, exercising decisive control over caste differentiation. The unique biochemical composition of royal jelly includes water, proteins, carbohydrates, lipids, vitamins, minerals, and numerous bioactive compounds, conferring antioxidant, anti-inflammatory, antimicrobial, and immunomodulatory properties beneficial to both honey bees and humans.
The main Royal Jelly Proteins (MRJPs) represent the most abundant protein fraction in royal jelly, constituting approximately 82-90% of its total protein content. These proteins are encoded by the MRJP multigene family, with nine distinct members (MRJP1-9) identified in the honey bee genome, predominantly located on chromosome 11. Within this family, the MRJP1 gene is particularly significant, as it encodes the most abundant royal jelly protein, royalactin, which functions as a primary factor directing larval development toward the queen caste and serves as a molecular marker for assessing royal jelly freshness and authenticity. The MRJP1 gene spans approximately 3038 base pairs, with six exons and five introns, and encodes a mature protein of 432 amino acids. Despite its well-established biological and economic importance, information regarding its genetic diversity remains limited, particularly within native Iranian honey bee populations (Apis mellifera meda). This investigation was designed to examine the MRJP1 gene polymorphism in Iranian honey bee populations and to evaluate potential associations with royal jelly production characteristics, aiming to assess existing genetic variation and to explore the use of MRJP1 as a candidate molecular marker for selecting superior royal jelly-producing colonies.
Method
The experimental population comprised 120 honey bee colonies maintained at an apiary in Bostanabad County, East Azerbaijan Province. Royal jelly production data were systematically recorded for each colony following harvest. For genetic analysis, worker pupae were collected and preserved in 70% ethanol solution before transport to the Genetics Laboratory at Urmia University. Genomic DNA extraction was performed using an optimized CTAB-based protocol, with DNA quantity and quality evaluated through NanoDrop spectrophotometry and electrophoresis on 1% agarose gels.
For molecular characterization, specific primers were designed to amplify a 443-base-pair fragment of the MRJP1 gene from the reference sequence. Primer sequences were forward: 5'-GATGCAACGCTGGTCATGTG-3' and reverse: 5'-TCACTTTGAGCGACGGTACG-3'. PCR amplification was conducted in 20 µL volumes using a commercial master mix, with thermal cycling including initial denaturation at 95°C for 5 minutes, 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute, and final extension at 72°C for 5 minutes. PCR products were evaluated on 1.5% agarose gels. Genetic polymorphism was investigated using Single-Strand Conformation Polymorphism (SSCP) analysis on 12% polyacrylamide gels with vertical electrophoresis at 330 volts for 5 hours.
Results
DNA extraction from all 120 samples yielded high-quality genomic DNA with concentrations exceeding 150 ng/µL. Purity evaluation revealed 260/280 ratios of approximately 1.8, indicating minimal protein contamination, and 260/230 ratios of approximately 2.0, confirming the absence of phenolic or carbohydrate contamination. PCR amplification of the 443 bp MRJP1 fragment was successful for all samples, with clear, sharp bands at the expected molecular weight and no non-specific products. SSCP analysis revealed remarkably uniform banding patterns across all 120 samples, with identical electrophoretic mobility and no evidence of conformational variants indicating sequence polymorphisms. This complete absence of variation demonstrates that the examined MRJP1 region is monomorphic within the studied Iranian honey bee population.
The principal finding is the complete absence of detectable polymorphism in the MRJP1 gene fragment examined. This contrasts with findings for other MRJP family members, in which substantial polymorphism has been documented in MRJP3, MRJP5, and MRJP8, whereas MRJP2 exhibits monomorphism similar to MRJP1. This heterogeneous pattern reflects distinct evolutionary mechanisms governing different members of this gene family. The pronounced conservation of MRJP1 can be attributed to purifying selection acting throughout the evolutionary history of honey bees. As the gene encoding the most abundant royal jelly protein, with established roles in caste determination and larval development, MRJP1 plays a critical biological role. Any non-synonymous mutations that alter the protein sequence would likely disrupt these essential functions, interfere with larval differentiation, and compromise colony social structure, and would be efficiently eliminated by natural selection. This interpretation is consistent with molecular evidence indicating MRJP1 conservation at nucleotide sequence, promoter architecture, and regulatory levels.
The essential functions of MRJP1 extend beyond caste differentiation to include contributions to the physical properties of royal jelly. The protein forms oligomeric complexes with apisimin, contributing to the characteristic high viscosity that enables royal jelly to support the development of queen larvae. Mutations disrupting protein structure would compromise these complex-formation capabilities, thereby providing additional selective pressure to maintain sequence integrity. Even without DNA sequence variation, phenotypic diversity could potentially arise through post-translational modifications. Research has documented that MRJP1 undergoes methylation, phosphorylation, and deamidation, generating multiple protein isoforms with potentially distinct functional properties without requiring underlying gene sequence variation. Such modifications may influence royal jelly characteristics while remaining invisible to DNA-based polymorphism screening. The sample size of 120 colonies, while substantial, may have been insufficient to detect rare polymorphisms present at very low frequencies. Investigations of larger European honey bee populations have identified polymorphic sites within MRJP1, demonstrating that variation at this locus is not absent across all populations. The presence or absence of polymorphism may depend on the specific population examined, geographic sampling scale, and gene regions targeted. Additionally, MRJP gene expression can be modulated by environmental and nutritional factors, suggesting that phenotypic variation in royal jelly production may arise from differences in gene expression regulation rather than structural variation. Even with a completely monomorphic MRJP1 sequence, variation in expression levels in response to environmental conditions could produce significant differences in royal jelly characteristics.
Conclusions
In the Iranian honey bee population studied, the MRJP1 gene appears to be highly conserved, with no detectable polymorphism in its coding region. This uniformity likely results from strong selective pressure on this locus, given the gene's crucial role in larval caste determination and the structural integrity of royal jelly. However, the lack of DNA-level variation does not imply a lack of functional diversity; post‑translational modifications and differences in gene expression levels may also give rise to phenotypic variation related to royal jelly production. Given the growing economic importance of royal jelly, further investigation of this gene in native Iranian populations seems necessary. Future research should focus on broader sampling, examination of promoter and intronic regions, study of post‑translational modifications, and analysis of gene expression under various environmental and nutritional conditions. Such an integrated approach would help clarify the molecular mechanisms of this gene and pave the way for marker‑assisted breeding programs.