Analysis On Expression Patterns And Prokaryotic Expression Of Pyruvate Dehydrogenase Kinase Gene Of Silkworms, Bombyx Mori

The silkworms (Bombyx mori) of bivoltine lay diapause eggs when they were incubated at 25℃under light, or lay non-diapause eggs when incubated at 15℃in darkness. Researches have revealed that the expression of pyruvate dehydrogenase kinase (PDK) in protein level is different under different incubation conditions of silkworm eggs, implying that BmPDK might play an important role in the mechanism of diapause in the silkworm. To study the function of BmPDK gene, the expression profile of BmPDK gene in different development stage and the influence of incubation conditions (temperature and lightness) during embryonic stage were analysed by using bivoltine silkworm variety Qiufeng as experimental material, and the cDNA of BmPDK gene was cloned and prokaryotic expressed.1. Analysis of structure and phylogenesis of BmPDK geneBmPDK gene structure and its protein were analysed by bioinformatics.The result indicates that the molecular weight of BmPDK protein is 46.92 kD, its isoelectric point is 6.61. There is no signal peptide sequence and transmembrane structure found. Scanty water area and hydrophilic groups of amino acid sequence are interlaced, the minimum of hydrophobic property is -2.5 and the maximum is 1.978. The N-terminal of BmPDK is mainly constituted byα-helixs and the C-terminal is composed byα-helixs andβ-foldes, in which fiveβ-foldes are arranged into a big folded face. N-terminal and C-terminal are connected byα-helix. BmPDK are closely related with Drosophila melanogaster, Aedes aegypti and Culex quinquefasciatus, and have far relationship with vertebrate.2,Expression profile of BmPDK of bivoltine silkworm and effect of incubation condition to expression regulationAdopted the method of half-egg batch laid by one moth of bivoltine silkworm variety Qiufeng, eggs were distributed into two groups of different incubation conditions, i.e. 25℃under light and 15℃in darkness. The experimental material were prepared at different development stages from full bulge embryo stage to adult, and the expression profile of BmPDK gene were detected on RNA level by Real-time PCR and data were analysed with SPSS 16.0. The results indicate the expression level of BmPDK is much higher than others at the periods of teachea colouring stage, head pigmentation stage, body pigmentation stage, first instar and adult stage, showing that BmPDK gene is very important in the last stage of embryonic development and the spawning season. The expression level of BmPDK gene increases enormously during the body pigmentation stage incubated in low temperature, and reduces enormously in high temperature group; in contrary, it increases enormously during first instar incubated in low temperature , and reduces enormously in high temperature group; till second instar, the expression level of BmPDK gene reduces to extremely low both in high and low temperature groups; in the ovaries of the pupation and adult stage, BmPDK expresses greatly in high temperature group. So, the influence of temperature on BmPDK expression mainly concentrates in body pigmentation stage, first instar,pupation and adult stage.3,Clone and prokaryotic expression of BmPDK gene cDNA of silkwormBy useing the total RNA of first instar of Qiufeng as template, the BmPDK gene was amplified and the products of PCR is connected to the cloning vector pMD-18T in order to construct the cloning plasmid pMD-18T-PDK. After cutting by EcoRI and Hindâ…¢, the gene is reconstructed in the prokaryotic expression vector pET-28a(+) and thus the prokaryotic expression plasmid pET-28a-PDK is constructed. pET-28a-PDK is transfered into E.coli BL21, which then being induced by IPTG and analyzed by SDS-PAGE in order to get the expression form of the target gene BmPDK as well as the best induction concentration and the best induction time. By using the Ni2+-NTA resin to purify the objective protein, the purified target protein is analyzed by Western blot to identify the purified BmPDK protein that gets. The enzymatic activity of expressed BmPDK is indirectly tested by adopting 2, 6-DCPIP. Enzyme cut electrophoresis and sequence results show that the whole cDNA of BmPDK is correctly expanded. Induced expression by IPTG, the SDS-PAGE result show that BmPDK protein is expressed in the form of inclusion body mostly. The best induced concentration is 0.8 mmol/L, and the best induced time is 8 hours. There is a 51KDa specific band in the picture of electrophoresis. After the purification of Ni²⁺-NTA column, an analysis of objective protein by Western blot with a result that the objective band is clear, shows that the establishment of prokaryotic expression vector pET-28a-PDK is successful, and the expression of objective band is correct. The expressive BmPDK is actived and apparent pseudo-first-order reaction rate constant k is 0.062.The results of this research supply experimental data for the further research of the function of the BmPDK gene and diapause mechanism, and make a basis for other relative follow-up study.