Proteomic-Based Approach to the Proteins Involved in 1-Deoxynojirimycin Accumulation in Silkworm Bombyx mori (Lepidoptera: Bombycidae)

Abstract

1-Deoxynojirimycin (DNJ) is the most abundant poly-hydroxylated alkaloid in the latex of mulberry leaves and it protects mulberry from insect predation. However, silkworms can survive the poisoning effect of DNJ and accumulate DNJ by consumption of the mulberry leaves. In order to determine the molecular mechanism of DNJ accumulation in silkworm, comparative proteomic analysis was employed to evaluate protein expression in two groups of silkworm bodies (the third instar silkworm bodies had the maximum content of DNJ throughout life, and the newly hatched silkworm bodies had no DNJ). Our results indicated some differentially expressed proteins in the third instar silkworm involved in material metabolism, energy metabolism, oxidation-reduction, detoxification, immune, and transport regulation may correspond to the accumulation of DNJ. Furthermore, the expression levels of five selected differentially expressed protein-encoding genes namely heat shock cognate protein (Hsp 70), glutathione S-transferase sigma 1 (GST), serine protease precursor (Ser), hemolymph protein (30K), and thiol peroxiredoxin (TPx) were investigated by quantitative real-time PCR and the accumulation of DNJ was measured by HPLC. Correlation analysis showed that the expression levels of Hsp70 and Ser were negatively correlated to DNJ accumulation with weak correlation, while 30K, GST, and TPx genes had positive correlation with DNJ accumulation. The findings suggested that these three proteins were probably important in the physiological process of DNJ accumulation in silkworm.

Fig. 1.

2-DE pattern of silkworm bodies proteins. The third instar silkworm bodies (A) and the newly hatched silkworm bodies (B). The identified differentially expressed protein spots in this study are indicated by arrows in the third instar silkworm bodies (A), the numbers corresponded to those are in Table 1.

Fig. 2.

Relative expression of selected five protein-encoding genes and DNJ content in silkworm bodies at different silkworm life stages. qRT-PCR technique was used to detect the relative expression levels of five genes, and HPLC was used to measure DNJ content in silkworm bodies from newly hatched to fourth-instar silkworm. (A: Hsp 70; B: GST; C: Ser; D:30K; E: TPx and F: DNJ content).

Fig. 3.

Correlation between DNJ content and the expression levels of selected five protein-encoding genes. (A: Hsp70-DNJ; B: GST-DNJ; C: Ser-DNJ; D: 30K-DNJ and E: TPx-DNJ).