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The context sequence motif surrounding the ATG initiation codon influences mRNA translation efficiency and affects protein production; however, the optimal sequence differs among species. To determine the optimal sequence for production of recombinant proteins in a transgenic silkworm, we compared 14-nucleotide context motifs around the ATG (ATG-context) in 50 silkworm genes and found the following consensus: (A/T)AN(A/T)ATCAAAatgN. We were also able to define the least-common motif: CCN(C/G)CGN(C/T/G)(G/C/T)(T/G)atgC, which served as a negative control. To examine the regulatory role of these motifs in protein expression, we constructed reporter plasmids containing different ATG-context motifs together with either the luciferase gene or an enhanced green fluorescent protein (EGFP) gene. These constructs were then used for comparison of luciferase reporter activity and EGFP production in BmN4 cells  in vitro  as well as in transgenic silkworms  in vivo . We detected 10-fold higher luciferase activity in BmN4 cells transfected with the consensus ATG-context motif construct, compared to the negative control plasmid. ELISA measurements of EGFP translation products with the corresponding constructs in BmN4 cells showed consistently similar results. Interestingly, the translation efficiency of the novel consensus ATG-context motif did not show the highest activity in the transgenic silkworms  in vivo , except for the fat body. The highest efficiency in the middle and posterior silk glands was produced by the sericin 1 context. Our results show that the ATG-context motifs differ among silkworm tissues. This result is important for the further improvement of the transgenic silkworm system for the production of recombinant proteins.

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Effect of ATG initiation codon context motifs on the efficiency of translation of mRNA derived from exogenous genes in the transgenic silkworm, Bombyx mori