Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx mori

Abstract

Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO₂ NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO₂ NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO₂ NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm.

Figure 1. Identification of TyrRS and SerRS PCR products and and recombinant plasmids using restriction enzyme digestion.

M: 250 bp DNA marker; 1–2: Positive controls of SerRS and TyrRS PCR products; 3–4: restriction enzyme digestion of SerRS and TyrRS PCR products; 5–6: 5′ UTR positive controls of SerRS and TyrRS; 7–8: restriction enzyme digestion of SerRS and TyrRS 5′ UTR; 9–10: 3′ UTR positive controls of SerRS and TyrRS; 11–12: restriction enzyme digestion of SerRS and TyrRS 3′ UTR.

Figure 2

Functional analysis of Structure domains of SerRS (A) and TyrRS (B).

Figure 3

Sequence comparison of SerRS (A) and TyrRS (B) from multi-species. The blackened residues indicate characteristic amino acids were potential species recognition sites.

Figure 4. Phylogenetic tree analysis of SerRS and TyrRS.

(A) Phylogenetic tree of SerRS, (B) Phylogenetic tree of TyrRS.

Figure 5. Specific expression of SerRS, TyrRS, AlaRS and GlyRS in silkworm tissues.

(A) Expression of SerRS, TyrRS, AlaRS and GlyRS in different tissues of silkworms. 1: silk gland; 2: midgut; 3: fat body; 4: Markov pipe; 5: Head; 6: gonads; 7: hemolymph. (B) Expression of SerRS, TyrRS, AlaRS and GlyRS in different parts of silk gland. 1: anterior silk gland, 2: middle silk glands; 3: posterior silk gland.

Figure 6. Effect of TiO₂ NPs treatment and phoxim treatment in silk gland.

(A) The component of silk protein in silk gland. (B) Transcription levels of SerRS, TyrRS, AlaRS and GlyRS in silk gland. 1: control; 2: TiO₂ NPs; 3: phoxim. (C) Fold change of free amino in silk gland. Treatments with different letters indicate significantly different values (*P < 0.05, **P < 0.01), Values represent means ± SD, n = 3.