Characterization and cDNA cloning of major royal jelly proteins of Apis cerana in Thailand

Abstract

The large subunit of the ribosomal (Ir) RNA gene of Thai hive bee, Apis cerana was PCR-amplified and sequenced. Based on these nucleotide sequences, genetic differentiation analysis of A. cerana was simplified to a PCR-RFLP based on Dra I. Four haplotypes of digested IrRNA gene were observed. Haplotype A was found in north, north-east and the central region whereas haplotype B was restricted to specimens from peninsular Thailand, Phuket, and Samui Islands. Haplotype C was found in 47.06% of A. cerana originating from Samui Island but was not found in other geographic samples. Geographic heterogeneity analysis and F[subscript ST] statistics indicated the existence of population differentiation of A. cerana in Thailand (P<0.0001). Analysis of molecular variance (AMOVA) also illustrated significant genetic differences between bees from the north-to-central region (A), peninsular Thailand & Phuket (B) and Samui Island (C) (P < 0.0001). RJ of A. cerana was produced from north to central and southern samples using a queen-rearing method. Yields of RJ productions were not significant different among different be population (P>0.05). Three families of AcMRJPs were chromatographically purified by Q-Sepharose followed by Sephadex G-200 columns. All purified AcMRJPs were glycoproteins. AcMRJP1 exhibited 2 native forms; monomer (50 kDa) and oligomer (300 kDa), with the pI of 5.2-5.7. AcMRJP2 was a 55 kDa protein with the pI of 7.0-8.2 whereas AcMRJP3 natively exhibited a dimeric of 115 kDa and a denatured monomeric form of 80 kDa with the pI of 7.4-8.4. The quantitative ratio of AcMRJP1 : AcMRJP2 : AcMRJP3 in RJ was 7.2 : 2.7 : 1. Under different stored condition (-20 ℃, 4 ℃ and 37 ℃ for 1-30 days), AcMRJP3 was less stable and easier degraded than AcMRJP1 and AcMRJP2. The cDNA library was established from hypopharyngeal gland mRNA. Four family of AcMRJPs and other important metabloic-related genes (α-glucosidase, glucose oxidase and apisimin) were identified. The full length AcMRJP1 was deduced and contained a 1299 bp ORF, encoding 433 amino acid residues with 48.3% essential amino acid. In addition, the complete AcMRJP3 transcript was isolated by RT-PCR. It contained 1824 bp ORF encoding 608 amino acid residues with 39.5% essential amino acid. Semi-quantitative PCR was successfully developed and verified that the expression level of these respective protein coding genes (AcMRJP1, AcMRJP2 and AcMRJP3) was in a ratio of 3.3 : 1.6 : 1 in hypopharyngeal glands of nurse bees. Expression of these genes was not found in newly emerged bees and AcMRJP1, AcMRJP2 and AcMRJP3 transcripts of nurse bees were 1.8, 2.5 and 2.0 times greater than those of forager bees.