Cloning and sequencing of cDNA encoding major royal jelly protein family 4 and 5 of Apis cerana in Thailand

Abstract

Royal jelly is the food for bee larvae and plays an essential role in honeybee nutrition. Major protein of royal jelly designated as MRJPs are member of one protein family consisted of 8 closely related proteins (MRJP1-MRJP8). In this study, MRJP3, MRJP4, MRJP5, and MRJP6 were isolated from hypopharyngeal gland of Apis cerana nurse bee by Reverse transcription-PCR (RT-PCR). The RT-PCR products were cloned, and then identified by restriction mapping and mucleotide sequencing. The partial nucleotide sequence of AcMRJP3 cDNA and complete mucleotide sequence of AcMRJP4, AcMRJP5 and AcMRJP6 cDNA were identified. Partial sequence of AcMRJP3 cDNA obtained as almost 100% identity in overlap sequence with the sequence of AcMRJP3 cDNA previously report. The nucleotide sequence at 3’ untranslated region (3’UTR) of AcMRJP3 gene was obtained in this study. The AcMRJP4 cDNA contained 1458 bp ORF encoding 485 amino acid residues with deduced molecular weight of 52.8 kDa, composed of 42.4% essential amino acids. The AcMRJP5 cDNA contained 1740 bp ORF encoding 579 amino acid residues with deduced molecular weight of 66.2 kDa, composed of 51.9% essential amino acids.The AcMRJP6 cDNA contained 1308bp ORF encoding 435 amino acid residues with deduced molecular weight of 47.4 kDa, composed of 46.8% essential amino acids. Nucleotide sequence of AcMRJP4, AcMRJP5 and AcMRJP6 cDNA showed high homology with those of AmMRJP4 (89%), AmMRJP5 (91%) and AmMRJP6 (92%), respectively. The deduced amino acids of AcMRJP5 showed the extensive repeat region located between amino acid residue position 367 and 540. The repeat unit was dominant in tripeptide DRM that occurred 51 times and located at the same position within A. dorsata and A. mellifera but differ in repeat unit sizes. The expression of MRJP4 cDNA as performed by using E. coli expression approach. The SDS-PAGE analysis revealed protein band of 53 kDa which was similar to the expected molecular weigh of 55.7 kDa estimated from the nucleotide sequence data.