Study On Bacillus Nematocida B16 Gene Knockout

We found that the deacetylase inhibitor nicotinamide (NAM) can improve the nematocidal activity of Bacillus nematocida B16significantly, it suggested that the process of B16nematicides was regulated by protein acetylation. In order to study on the regulatory mechanism, it is necessary to knock out some related genes. But it is very difficult to knock out genes in B16, so we explored several methods of knocking out gene in B16in my thesis. First, we found the acetylase gene acuA and deacetylase gene acuC by DNA and protein sequence blast, and cloned these two genes. Second, we screened out a rifampicin resistant strain of B16named B16Rif" by ultraviolet irradiation. In this mutant strain, we improved the transformation efficiency of plasmid into B16by conjugative transfer. On this basis, we reconstructed the conventional integrating vector pCP115, the temperature-sensitive vector pKVM2and the CRISPER/Cas9system vector pCas9, giving them the ability of transconjunction. Last, we used these three vectors to construct knock-out vectors of acuC gene. Then, we transformed these vectors into B16Rif+by conjugative transfer, selected transformants and conform the knock-out of acuC gene by clone PCR. Unfortunately,no positive transformants was screened out, it meant that the acuC gene was not been knocking out. In this thesis,we have found that nicotinamide can improve the nematocidal activity of B16observably, and this result suggested protein acetylation participated in the process of B16nematicides. In addition, we solved the problem that the transformation efficiency of plasmid into B16is very low. Although we explored a variety of ways to knock out genes in B16Rif+, the target gene had not been knocked out. This study lays a foundation to establish an efficient method of gene knock-out and to clarify the molecular mechanism of protein acetylation in regulating nematicidal process of B16.The main results of this study:1. NAM improve the nematocidal activity of B16We added deacetylase inhibitor nicotinamide NAM into the midium which cultivate B16and detect the nematocidal activity. Then we found that the activity increased from38%to93%, it means that NAM can drastically enhance the nematocidal activity of B16. This result suggested that protein acetylation participated in the nematicidal process of B16.2. Transconjunction increased the transformation efficiency of plasmid into B16By blast, using the sequence of Bacillus subtilis, we found the homologous proteins of acetylase AcuA and deacetylase acuC and srtN in B16. The identity of the former is85.7%and the latter is81.4%and77.7%. We utilized the integrated plasmid pCP115to construct a deacetylase gene knock-out vector pCP115-acuC, and then we transformed this vector into wild-type B16by means of chemical transformation. Unfortunately, we did not get any transformant after several attempts. This shows that the transformation efficiency of plasmid is very low.By mutagenesis of ultraviolet radiation, we got three rifampicin resistant strain of B16named B16Rif+. In one of the mutant strain, we use a transconjunctive plasmid pRK415-kan to test the transformation efficiency, and found this plasmid can transform into B16Rif+with high efficiency. Up to now, we eventually solved the problem of low efficiency of chemical transformation.3. Reconstructed the plasmid pCP115to be a transconjunctive plasmid and constructed a acuC gene knock-out vectorWe reconstructed the plasmid pCP115, including the insertion of the gene mob from plasmid pSUP202and the kanamycin gene kan. All this made it to be a transconjunction shuttle vector with resistance of kanamycin. On this basis, we constructed a acuC gene knock-out vector pCP115-mob-kan-acuC, then we transformed it into B16if+by transconjunction. We screened transformants on the double resistance plate of kanamycin and rifampicin. After conformation by clone PCR, we still did not get any positive transformant, and the vector did not integrate into the genome of B16Rif+4. Utilize the temperature-sensitive vector pKVM2to knock out the acuC geneTo solve the problem that vector pCP115-mob-kan-acuC did not integrate into the genome, we reconstructed the plasmid pKVM2by inserting the kanamycin gene kan. Then, we constructed a knock-out vector of acuC gene, named pKVM2-kan-acuC. And transformed it into B16Rif+by transconjunction. We screened transformants on the double resistance plate of kanamycin and rifampicin. And select one transformant to do a temperature screening from42℃to46℃. The sesult of clone PCR suggested that the vector did not integrate into the genome of B16Rif+. It meant that the acuC gene was not been knocked out.5.Try to knock out the acuC gene by utilizing the CRISPER/Cas9systemBy using the plasmid pCP115-mob-kan as a backbone,a fragment about5500bp from the plasmid pCas9, which include Cas9enzyme gene、tracrRNA and crRNA, was inserted into pCP115-mob-kan.Then we got a transconjunction vector pCP115-mob-kan-Cas9which has a kanamycin resistance. Next step, We plan to construct a acuC gene knock-out vector pCP115-mob-kan-Cas9-acuC to knock out the acuC gene in B16Rif+. In the interest of time, we did not finish this work.