Analysis Of The Characteristics Of Protein And Gene Expression In Fat Body Of Bombyx Mori After The Induction Of Organophosphorus Pesticide

In recent years, along with the human genome project smoothly conducting and finishing, the research of life science enter into the post-genome era. In this era, the main study object of the life science research has gradually developed from genomics to the functional genomics, proteomics and cell metabolomics, etc. Recently, proteomic analysis technology has been used extensively in biological innovative researches and has become one of the most essential technologies in functional genomics. The proteomics is the study of various protein properties (expression level, post-translation modification, etc) at the whole cell level ever whole body to obtain a global physiological and biochemical processes of cells and regulatory networks at the protein level. Proteomic technology has also been applied to analyze simultaneously hundreds or even thousands of protein. As the organic phosphorus insecticides for pest control of crops and human life in the wide range of applications, leading to a large number of insect resistance to them. Bombyx mori is a model organism of Lepidoptera insect. As silkworm and lepidoptera pests are similar in physiological and biochemical and mechanism of pesticide metabolize. Study on the resistance of silkworm to organic phosphorus will provide certain theoretical basis for the prevention and control of Lepidoptera pests and breeding of resistant variety of silkworm.In this study, we measured the toxicity of Phoxim on Bombyx mori. To study the resistance mechanism of ognanophosphorus of Bombyx mori at the protein level, high resolution, two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption ionization time of flight mass spectromet (MALDI-TOF MS) was applied to identify differences in protein. And then we studied the gene transcription level of the different protein by semi-quantitative method. The main results we obtained were as follows:1 The LC50 of male and female Bombyx mori to the phoxim.To determine the LC50 of different sex of Bombyx mori, we treated the male and female silkworm with different concentrations of phoxim. The results showed that the LC50 of female silkworm was in 3.932~4.184μg/mL and the LC50 of male silkworm was in 3.989~4.219μg/mL, the resistance of the male silkworm was slightly higher than the female ones. The results provided a theoretical basis for the materials of the following work.2 Protein expression profiling of fat body in male Bombyx mori induced by ognanophosphorus.2.1 The establishment of patterns of fat body protein in male Bombyx mori induced by ognanophosphorus using two dimensional polyacrylamide gel electrophoresis.We compared the different proteins of fat body expressed in male silkworm induced by ognanophosphorus using two-dimensional electrophoresis technology. The results of ImageMaster 6.0 analysis showed that 465 protein spots were detected in the control group, while 396 protein spots in the experimental group, and the matches were 348. Comparative analysis showed that the matched value between the two groups were 73.54%. Protein spots were mainly distributed in the molecular mass of 20.1~66.4 kD and the pI of 5~8.2.2 The spectrum identification of different proteins in male Bombyx mori fat body induced by ognanophosphorus.The interested protein spots were identified by MALDI-TOF MS. The proteins identified were phosphoserine aminotransferase(SerB) and 5'-methylthioadenosine phosphorylase(MTAP), respectively. These proteins were related to different metabolic pathways, such as serine biosynthesis, serine metabolism, glycolysis, gluconeogenesis, nucleotide metabolic pathways, and so on.2.3 The enolase gene transcription activity of 5 instar larvae Bombyx mori induced by ognanophosphorus.Inducement effect of ognanophosphorus treatment on transcription level of SerB gene in the 5th instar larvae was determined in six tissues: brain, midgut, silk gland, fat body, testis and malpighian tubules by semi-quantitative RT-PCR method, respectively. The result showed that SerB gene expression increased in six tissues after induced by Phoxim and the specific value is 183%, 4.48%, 9.55%, 2.6%, 1.88% and 15.25%, respectively. Both gene expression and protein expression of SerB in fat body showed an upward trend.3 Protein expression profiling of fat body in female Bombyx mori induced by ognanophosphorus.3.1 The establishment of patterns of fat body protein in female Bombyx mori induced by ognanophosphorus using two dimensional polyacrylamide gel electrophoresis. We compared the different proteins of fat body in female silkworm induced by ognanophosphorus using two-dimensional electrophoresis technology. The result of ImageMaster 6.0 analysis showed that 516 protein spots were detected in the control group, while 457 protein spots in the experimental group, and the matches were 409. Comparative analysis showed that the matched value between the two groups were 70.25%. Protein spots were mainly distributed in the molecular mass of 20.1~66.4 kDa and the pI of 5~8. 3.2 The spectrum identification of different protein in female Bombyx mori fat body induced by ognanophosphorus.The interested protein spots were identified by MALDI-TOF MS. The identified proteins were silkworm glyceraldehyde phosphate isomerase/triosephosphate isomerase, silkworm heat shock protein, actin, cytochrome P-450 c21 hydroxylase, trypsin/insulin, betaine/carnitine/choline ABC superfamily ATP binding transporter protein, unknown protein, etc. These proteins were related to different metabolic pathways, such as protein folding, respiratory chain reaction, glycolysis channels, microtubule assembly, and so on. These proteins related to different metabolic pathways may be related to silkworm resistance-related.Bombyx mori belongs to Lepidoptera Saturniidae insect. Many scholars at home and abroad have conducted a large number of research on silkworm pesticide resistance, but few from the level of proteomics research of silkworm resistance to organophosphorus pesticide and silkworm resistance to organophosphate pesticide-related protein has not been reported. The preliminary identification of the characteristics of the 4 proteins in silkworm fat body after the induction of the organic phosphorus pesticide, suggesting that these proteins may play a certain role in some part of the process of silkworm metabolic detoxification of organophosphorus pesticides. The results of this study to research the sensitivity of silkworm chemical pesticides was of important significance for the resistance, laid a theoretical foundation for the resistance of silkworm breeding and development of efficient insecticides against Lepidoptera pests and provided some theoretical basis for the prevention and control of the pest lepidoptera.