Cloning And Expression Of Biocontrol Genes From Lysobacter Enzymogenes Strain OH11

Control of plant diseases which caused by fungi, bacterium and nematode is a key problem in agricultural production. Many diseases control mainly depend on the chemical pesticide. Using too much chemical pesticide had resulted in pollution in the environment and did harm to human being and domestic animals. Meanwhile, it induced resistance of pathogen to chemical pesticide. But biological control is safe to human being and domestic animals, has little pollution in the environment, sometimes can control some pest for a long time, and easy to operate. So biocontrol has been focused recent years.Lysobacter enzymogenes strain OH11 is a biocontrol agent isolated from soil, and has biological control to fungi and bacterium. It is characterized by a high GC content, gliding motility and can produces many extracelluar enzymes such as chitinase,β-1, 3-glucanase, a-lytic protease and so on. While enzymatic lysis has been implicated as one mechanism of biocontrol by bacterial agents.We analyse the nucleic acid sequences of biocontrol genes from Lysobacter enzymogenes strain C3 and N4-7, and design the primers. PCR amplification the chiA gene,gluA gene,gluC gene and a-lytic protease gene from strain OH11. The genes were cloned to the expression vector pET21a(+) or pET30a(+). After that we select the positive clones and named BLChiA,BLGluA,BLGluC,BLaLP and sequenced. In this study, a homolog of chiA was cloned from L. enzymogenes strain OH11, which shares 97.24% amino acid identity (96.85% similarity) with strain C3 and 95.93% amino acid identity (95.74% similarity) of strain N4-7. A homolog of gluA was cloned from strain OH11 which shares 98.06% amino acid identity (96.77% similarity) with strain C3 and 94.57% amino acid identity (94.57% similarity) of strain N4-7. A homolog of gluC was cloned from strain OH11 which shares 97.42% amino acid identity (96.99% similarity) with strain C3 and 96.12% amino acid identity (95% similarity) of strain N4-7. A homolog of a-lytic protease gene was cloned from strain OH11, which shares 90.77% amino acid identity (92.93% similarity) with strain ATCC 29487 and 91.02% amino acid identity (92.68% similarity) of strain ATCC 29478. The expression strains were induced by IPTG and test by 15% SDS-PAGE, then over-cultivated and fragmentation by stress, proteins were purified by Ni²⁺ colum. The purification protein can hydrolyse the substracts and suppress the mycelium of Rhizoctonia solani.