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Analysis on Expression Patterns and Prokaryotic Expression of Pyruvate Dehydrogenase Kinase Gene of Silkworms, Bombyx Mori

Author: SongHaiZuo
Tutor: ShenXingJia
School: Jiangsu University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Silkworm Incubation conditions Pyruvate dehydrogenase kinase Real-time PCR The prokaryotic expression
CLC: S881.24
Type: Master's thesis
Year: 2011
Downloads: 16
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Abstract

Bivoltine breeds of silkworm (Bombyx mori) the silkworm eggs high temperature light incubation diapause eggs and non-diapause eggs the cryogenic dark Incubation production. Studies have shown that the silkworm eggs under different incubation conditions pyruvate dehydrogenase kinase (PDK) differential expression at the protein level, this implies BmPDK the silkworm diapause mechanism may play an important role. To this end, the paper in order to the bivoltine silkworm varieties Qiufeng \expressed. Silkworm PDK gene structure and molecular evolution analysis using bioinformatics methods of the BmPDK gene and its protein structure analysis Show BmPDK protein molecular weight of 46.92 kD, an isoelectric point of 6.61, no signal peptide sequence and transmembrane structure, the amino acid sequence of the hydrophobic and hydrophilic regions staggered, the hydrophobicity of the minimum value is -2.5, and the maximum is 1.978. BmPDK the N-terminal portion is mainly composed of α-helix; the C end portion folding two structures of the α-helix and β, wherein five β-sheets arranged to constitute a large folding face the N-terminal part and the C-terminal portion is connected through the α-helix. The silkworm BmPDK and Drosophila, Aedes aegypti and Culex Culex and other insects close genetic relationship, while relatively distant kinship with vertebrate. Silkworm bivoltine varieties PDK gene expression profiles of silkworm egg incubation conditions on the expression of moth half-points method bivoltine varieties Qiufeng silkworm eggs were divided into 2 groups, respectively, with a high temperature (25 ° C) The dark incubation light and low temperature (15 ℃), respectively in amyl an embryonic stage to the moth of the different developmental stages of preparation of the experimental material, using real-time PCR method analysis BmPDK differential expression at the RNA level, and using SPSS16.0 software on data for statistical analysis. The results showed BmPDK the already 3, has ₄, have ₅, hatched silkworm moth period expression level was significantly higher than in other periods, indicating BmPDK an important role in the late embryonic development and spawning. Low temperature incubation of expression BmPDK has five sharp rise in the high temperature dropped significantly; hatched silkworm period expression levels increased substantially low temperature incubation, the high temperatures dropped significantly; instar, high temperature incubation and low temperature catalytic Green expression were reduced to very low; the pupal stage ovarian and moth period, high temperature incubation BmPDK also a large number of expression. Therefore, the the incubation of temperature on BmPDK expression of mainly has five, ants silkworm pupae and moth period. 3, the silkworm BmPDK cDNA cloning and prokaryotic expression hatched silkworm with \Via EcoRI and Hind Ⅲ digested and then the recombinant prokaryotic expression vector pET-28a () to construct a prokaryotic expression plasmid pET-28a-PDK. PET-28a-PDK was transformed into E. coli BL21 by IPTG induction and SDS-PAGE analysis of the purpose the protein BmPDK the expressions optimal induction concentration, optimal induction time. The target protein was purified using Ni ^{2 -NTA resin, using Western blot analysis of protein purification purposes, in order to identify the resulting purified BmPDK protein, and finally with 2,6 - dichloro indole phenol method indirect expression activity of BmPDK. The sequence of the PCR and sequencing showed that the the amplified BmPDK gene is correct. After IPTG induction, SDS-PAGE electrophoresis showed the BmPDK protein was expressed as inclusion bodies, IPTG was best induced concentration of 0.8 mmol / L, and the optimal induction time is 8 hours, visible on the electropherogram about 51KDa specific fusion protein band. Ni2-NTA column purification of the target protein by Western blot analysis, the results show that the target band, no hybridization with successfully constructed prokaryotic expression vector pET-28a-PDK purpose strip properly express. The expression BmPDK activity apparent first order reaction rate constant k = 0.062. The results of this study for further research the silkworm BmPDK gene function and diapause mechanism provides experimental data, but also laid the foundation for other follow-up study.}

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Sericulture > Silkworm basic science > Silkworm physiology, genetics,ecology, biology,physics, biochemistry > Silkworm biochemistry
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