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Regulation and Expression of gab Gene Cluster in Bacillus Thuringiensis

Author: WangWei
Tutor: GaoJiGuo
School: Northeast Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Bacillus thuringiensis γ-aminobutyric acid metabolism bypass γ-aminobutyric acid Succinic semialdehyde gab gene cluster sigL gene Insecticidal Crystal Proteins
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 21
Quote: 0
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Abstract


Bacillus thuringiensis (Bacillus thuringiensis, referred to as Bt) is the most widely used insecticide in the field of agricultural biological control microorganisms. The most important feature of the Bt Unlike other Bacillus, Bt in the form spores, which can create efficient insecticidal activity of parasporal crystal protein. γ-amino butyric acid (γ-aminobutyric acid, GABA; GABA shunt) is widely present in the biological bypass vivo gab gene cluster related BT GABA shunt gabT the gene and gabD of genes encode the γ-aminobutyric acid transaminase ( GABA transaminase, GABASE) and succinic semialdehyde dehydrogenase (Succinic semialdhyde dehydrogenase, SSADH), respectively catalytic GABA and succinic semialdehyde (Succinic semialdhyde, SSA) is generated to generate succinic acid, and SSA enter the Krebs cycle. Early in the research group, the Institute found gab gene cluster in Bt under σL factor control and positive regulation by regulation of this gene cluster the gene gabR coding the product GabR protein. Transcription of the gene cluster the GABA shunt product for Bt gab impact has been reported. In this paper, the main column studies on Bt gab gene cluster transcription of its mechanisms, as well as the GABA shunt block and σL factor of spores and crystals around the GABA shunt core product of GABA and SSA. The analysis showed that the β-galactosidase activity, GABA and SSA could significantly induce transcriptional activity of gabT gene promoter, which SSA induced phenomenon more evident than GABA. Either GABA or SSA can not the induction gabR gene the promoter and sigL gene promoter transcription activity. HD (△ gabT) the HD (△ gabR), HD (△ gabD) and HD (△ sigL), into gabT gene promoter and lacZ fusion expression plasmid, respectively, in the four mutant gabT, gabR, gabD and sigL analysis of each of the mutant β-galactosidase enzyme activity in the culture medium were added to GABA and SSA results show that when Add GABA the HD (△ gabT), the HD (△ gabD) of gabT gene promoter transcription activity are high in the original strain the HD73; while adding succinic semialdehyde the HD (△ gabD) the gabT gene promoter transcriptional activity was significantly higher than the transcriptional activity of the the the HD73 and HD (△ gabT), gabT gene promoter, the above results indicate that the bacteria in vivo GABA and SSA accumulation induced transcriptional activity for gabT gene promoter. The results show that GABA and SSA for the HD (△ gabR) and HD (△ sigL), promoter gabT gene transcriptional activity induced complete loss, proved that this inducing activity is also a by σL factor control, and by GabR positive regulation. By the RT-PCR results show that, gabT the gene and gabD of the gene of the transcription of the GABA and SSA is determined by the 5'RACE method gabT gene transcription initiation site. The the HD73 strain of Bacillus thuringiensis the cloned gabR and sigL gene prokaryotic expression. These two gene expression vector, pET-21b, and the pGEX-4T-1 vector were constructed and transformed into E. coli BL21 (DE3) strain, SDS-PAGE showed that both genes are expressed respectively, wherein the obtained a the purified protein GabR. Laid the foundation for the further study of the molecular mechanism of the gab gene cluster. On the other hand this study based on Bacillus thuringiensis HD-73 strains and mutant strains analyzed the GABA shunt blocking the lack of impact on the of Bt growth capacity and ability of spore and sigL gene function. The results show that, GABA shunt blocking no significant effect on the growth and production of crystal protein of Bt ability, has resulted in a decline in Bt-producing Bacillus ability GABA shunt with Bt spores formed certain. The results of the growth curve, in relatively barren nutrition SM medium the HD73 with HD (△ sigL) growth difference is small, but in a nutrient-rich LB medium can be observed between the two significant differences. SDS-PAGE analysis results show that the The sigL gene function deletion significantly reduced the yield of the Cry proteins, and the β-galactosidase enzyme activity analysis proved that this difference in the amount of expression is not a transcriptional level caused on. The above results proved that some of sigL gene loss-of-function and its regulation of these metabolic pathways and Bt growth and spores and crystal protein closely related to the production of.

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