Cloning And Studying On Inhibitory Activities Of FabH Gene Of Escherichia Coli And Xanthomonas Oryzae Pv. Oryzae

Bacterial blight(BB)is a destructive disease caused by the plantpathogenic bacterium Xanthomonas oryzae pv.oryzae.It is one of the most serious bacterial diseases of rice that can result in a huge production loss of rice from 10%to 50%,or even worse.To date,no effective drugs have been developed against this disease and it is essential to find a drug against Xoo in order to halt rice-production losses.?-ketoacyl-acyl carrier protein synthase?[(FabH)initiates chain elongation in type II fatty-acid synthesis and is also an essential enzyme for bacterial viability,which makes it become a popular target for new antibacterial drugs.It is essential to understand characteristics and reaction mechanism of FabH,in order to develop a kind of new antibacterial agents with FabH inhibitory activity.In this study we designed a pair of primers according to the fabD and fabH gene sequence of E.coli K12 MG1655 and Xoo KACC10331,which had published in GenBank,and then amplified the whole gene by PCR.We obtained the correct fragment by PCR and cloned the fragment into pET-28a(+)expression vector.To make sure recombinant plasmid pET-28a-fabHEc,pET-28a-fabDEc,pET-28a-fabHXoo,pET-28a-fabDXoo,was constructed correctly,we digested the plasmid with restriction enzymes and sequenced the fragment.Then we transformed right recombinant plasmid into E.coli BL21(DE3)competent cells.The protein encoded by the plasmid that transformed into the bacteria.SDS-PAGE analysis proved the recombinant plasmid could express the right fusion protein.Afterly,we comparised FabHXoo's amino acid sequence with FabHEc's,then we found that its similarity is 45 percent with the same catalytic sites.The catalytic triplets were both Cys112-His244-Asn274.It illustrated that inhibitors against FabHEc's catalytic sites had greatest probably inhibition on FabHXoo.Lastly,we constructed two catalytic reaction of E.coli FabH and Xoo FabH in vitro by using FabHEc,FabDEc,FabHXoo,FabDXoo,proteins.We added the holo-ACP,malonyl-ACP,acyl-CoA and other cofactors to keep on research about some compounds against with E.coli FabH and Xoo FabH,suggesting that 4,5-Dichloro-[l,2]dithiol-3-one had some inhibition on Xoo FabH.We also studied their antibacterial activities against Xoo,and showed it was the potent antibacterial activity with MIC value of 2?g/mL against Xoo.This provided valuable information for the design of antibacterial agents for subsequent test.To research if the FabH played a key role in the growth of bacteria,we used homologous double exchange technology to knockout the target gene fabH of Xoo KACC10331.Through comparing the growth activity of the differences between mutant and wild type,we found that mutant strain grow at a slower pace,explained that the fabH is a necessary gene for Xoo.