Differential Proteomic Analysis And Research On DNA Methylation Transferase Expression Of Silkworm Moultinism Variation

Silkworm moultinism determined by the major genes on autosomes, as well as under the regulation of thoses genes associated with sexual maturity. Silkworm moultinism are vulnerable to the external environment, such as incubation temperature, the larvae of the nutritional conditions. Besides, it can also be changed by the concentration alteration of juvenile hormone and ecdysone. In order to discuss the molecular mechanism of silkworm moultinism changed by environmental factors, we used two-dimensional electrophoresis and electrospray ionization technology to analysis the differential protein expression in hemolymph and midgut tissue between normal silkworm and moultinism variated individuals before and after the last molting. We also detected the gene expression level of DNA methylation transferase using fluorescent real-time quantitative PCR technique to explore the relationship between moultinism variation and DNA methylation. The results are as follows:(1) In this paper, we studied the impact of moultinism on trimolter variety"Sanmian A"and"Bai-Yu C?3A"and tetramolter variety"D92 yellowish green"after deal with different incubation temperature (high temperature 28℃and low temperature 20℃). We discovered that there was a high tetramolter generation rate in high incubation temperature treatment groups, while hardly tetramolter generation rate in low incubation temperature treatment groups. But incubation temperature had little influence to tetramolter"D92 yellowish green". Further investigate on the offspring moultinism of tetramolter individuals of trimolter variety showed that the vast majority of the offsprings recovered trimolt character. (2) To study the molecular mechanism of silkworm moultinism alteration induced by environmental factors, the silkworm varieties of four-molter"898 yellowish green","D92 yellowish green"and trimolter"Sanmian A"were used as the materials. By control the environment condition, individuals of different moultinism were induced. With the techniques of two-dimension electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI- TOF MS), the expression differences of hemolymph and midgut tissue proteins between the normal and moultinism altered larvae of the silkworm before and after the last molting were analyzed. Totally 13 proteins up or down expressed were detected including transferrin, chymotrypsin inhibitor (CI), polyadenylate binding protein, 27kDa glycoprotein, ribosomal protein and mitochondrial ribosomal etc. The expressional variations of those proteins might relate to the metabolic level of juvenile hormone and ecdysone in the silkworm larvae. The alteration of moultinism may be a physiological reaction to the environmental changes. We found the FK506-binding protein, actin-depolymerizing factor and profilin had different expression level between moultinism variation and normal silkworm in midgut tissue.(3) Using fluorescent real-time quantitative PCR technique, we studied the difference of BmDnmt2 gene expression between moultinism vatiations and normal silkworm to explore the relationship between silkworm moultinism and DNA methylation. We discovered that BmDnmt2 gene expression level in hemolymph was almost no difference. But in the head of 898 yellowish green silkworms, the BmDnmt2 gene expression level in moultinism vatiation is lower than normal silkworm, only 0.8 of normal silkworm during the last molting while 0.75 during during the beginning of the last instar. It indicated that the methylation of some genes may reduce, which may influence silkworm moultinism.