Transcriptional Regulation Of Genes Related To MEP Metabolic Pathway In Bacillus Thuringiensis

Bacillus thuringiensis?Bacillus thuringensis,Bt?,a gram-positive bacterium,produces parasporal crystals with insecticidal activity against a variety of agricultural and forestry pests at the same time.It is the most widely used microbial insecticide.Terpenoids,widely distributed in the biological field,are important living substances.At present,there are two biosynthetic pathways of terpenoids,namely methylvalerate?MVA?pathway and 2-methyl-D-erythritol-4-phosphate?MEP?pathway.There are eight genes in Bt that encode enzymes related to MEP metabolism pathway.In this study,the transcriptional regulation of these gene was studied.The following results have been achieved:Bioinformatics analysis found that eight genes in Bt encode the enzymes associated with the MEP metabolic pathway,thedxs gene?HD73₄479?encoding1-deoxy-D-xylulose 5-phosphate synthase,the dxr gene?HD73₃605/dxr1 and HD73₄104/dxr2?encodes 1-deoxy-D-xylulose-5-phosphate reduction isomerase,the isp D?HD73₀085?gene encodes 2-C-methyl-D-erythritol-4-phosphotransferase,theisp E?HD73₀043?gene encodes 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase,the isp F?HD73₀086?gene encoding 2-C-methyl D-erythritol-2,4-diphosphate synthase,theisp G?HD73₄584?gene coding 4-hydroxy-3-methyl-2-alkenyl diphosphate synthase,theisp H?HD73₄593?gene encoding4-hydroxy-3-methylbut-2-enyl diphosphate reductase,the fni?HD73₁728?gene encodes isoamylene diphosphate isomerase.The isp D and isp F genes are in one gene cluster.These genes have homologous genes in Bacillus subtilis.By RT-PCR and 5 'RACE experiments,it was determined that the dxs gene and its downstream gene formed a transcription unit,and that the transcription initiation site?TSS?was located at the G base,which is at 133 bp upstream of theinitiation codon GTG of dxs.The dxr1?HD73₃605?gene forms a single transcription unit,and TSS is located at the G base,which is at 39 bp upstream of the initiation codon ATG of dxr1.The dxr2?HD73₄104?gene forms a transcription unit with nine genes upstream and downstream.TSS is located at the G base of the upstream codon ATG of the first gene?HD73₄110?in the cluster.The gene clusters of isp D and isp F are composed of five genes to form a transcription unit.TSS is located in the G base of the upstream,which is at 11 bp of the first gene?HD73₀084?start codon in the gene cluster.The isp E gene forms a transcription unit with three of its upstream and downstream genes,and TSS is located at the G base,which is at 138 bpupstream of the first gene?HD73₀041?start codon ATG in the cluster.The gene cluster of isp G gene is composed of three genes to form a transcription unit.TSS is located in the A base,which is at 25 bp upstream of ATG of the first gene?HD73₄582?in the gene cluster.The isp H gene forms a transcription unit with its upstream gene.TSS is located at the A base,which is at 24 bp upstream ofthe first gene?HD73₄592?start codon ATG in the cluster.The fni gene forms a transcription unit with its upstream gene,and TSS is located at the C base,which is at 76 bp upstream of ATG,the start codon of the first gene?HD73₁727?in the cluster.It was constructed that the expression vector of the lac Z reporter gene of the promoter about the relevant gene of the MEP metabolic pathway,and the activity of the yeast-galactooligosaccharide was determined in the Bt HD73 strain.The results showed that in SSM culture medium,?-galactosidase activity was determined from T1 to T8,these promoters have transcriptional activity.The transcriptional activity analysis showed that the transcription of dxr1 gene was controlled by Sig H.The deletion of dxr1 or dxr2 gene had no significant effect on cell growth,spores formation rate and Cry1 Ac protein yield,but decreased DXR activity.