Cloning, expression and genomic organization of major royal jelly proteins 1 and 2 genes of the honey bee Apis cerana

Abstract

Major Royal Jelly Protein (AcMRJP) cDNAs of Apis cerana were isolated from head of Apis cerana nurse bee by Reverse transcription-PCR (RT-PCR). The open reading frames (ORFs) of AcMRJP 1 and AcMRJP2 were 1,302 and 1,392 bp encoding 433 and 463 amino acid residues protein, respectively. Nucleotide sequence of AcMRJP 1 and AcMRJP2 cDNA showed high homology with those of AmMRJPl (93%) and AmMRJP2 (92%), respectively. Deduced amino acids showed high essential amino acid content of AcMRJP 1 and AcMRJP2 (47.4% and 45%, respectively). The genomic organization of AcMRJP 1 and AcMRJP2 genes were determined by PCR. The AcMRJP 1 and AcMRJP2 gene sequence spans over 3,663 bp and 3,963 bp, respectively. Both AcMRJP 1 and AcMRJP2 genes contain six exons separated by five introns. All intron-exon boundaries followed the GT/AG rule. Sequence analysis of the 5’ upstream regions revealed a putative TATA-box, locating approximately 31-32 bps upstream of the predicted transcription start sites of each gene* The presence of potential recognition sequences for ultraspiracle (USP) transcription factors were observed. The AcMRJP1 and AcMRJP2 cDNAs were cloned into expression vectors for expression in E. coli. SDS-PAGE analysis revealed protein band of 50 and 55 kDa corresponding to the expected molecular weight of approximately 47.9 kDa and 51.7 kDa for AcMRJP 1 and AcMRJP2, respectively. The expression of AcMRJP 1 and AcMRJP2 in E. coli was maximal at 4 hours after IPTG induction. The AcMRJP 1 and AcMRJP2 were expressed as inclusion body and purified using affinity chromatography. These bands were eluted with 250 mM imidazole and confirmed by N-terminal sequencing and Western blot analysis. The expressed recombinant plasmids containing AcMRJP 1 cDNA were constructed and introduced into potato (Solanum tuberosum L.) and rice (Oryza sativa L.) using Agrobacterium-mediated transformation method. The constructed recombinant plasmid for rice, AcMRJP 1 cDNA was inserted under the control of the cauliflower mosaic virus 35S promoter whereas for potato the cDNA was inserted under the control of the cauliflower mosaic virus 35S, or granule bound starch synthase (GBSS) or patatin B33 promoter. Successful expression of AcMRJPl mRNA and AcMRJPl in both potato and rice were obtained when all four recombinant plasmids were transformed as analyzed by RT-PCR and Western blot analysis.