Construction Of Biocontrol Agent Constitutively Expressing Harpin Via Type â…¡ Secretion Pathway

The application of genetic engineering to develop biocontrol bacteria is a promising measure for control of plant diseases. In this research, a protein named Harpin, which is the elicitor that can cause hypersensitive response, and simultaneously induce plant resistance to diseases, secreted via the sec-independent type â…¡ secretion pathway, was expressed and secreted in E coli. via the sec-dependent type â…¢ secretion system. The secretion-expression vector pExSec1 which needs induction was used in the experiments. To reduce the induction cost, the gene that encodes this fusion protein was cloned into the constitutive-expression vector pKK233-2. The new plasmid was transformed into a plant epiphyte P. agglomerans 308R which has been used for biocontrol. We studied the secretion and expression, the hypersensitive response activity (HRA), and the genetic stability of this recombinant biocontrol bacterium.Firstly, structural gene hrpN in plasmid pCPP430, which carries the hrp gene cluster, was ligated with pGEM-T vector to get plasmid pGEM-T-hrpN. Through restriction enzyme digestion, the isolated hrpN gene was cloned into the secretion-expression vector pExSec1. The new plasmid pExSec1-hrpN was transformed into E coli. BL21 (DE3) by CaCI2 chemical transformation method. The results showed that after the induction with IPTG, SZZ::Harpin fusion protein was detected both in CFEP and culture supernatant. The fusion protein was not only secreted and expressed via type â…¡ secretion system, but also had the HRA.Secondly, the PCR product of the gene that encodes SZZ::Harpin protein, using pGEM-T vector, was cloned into the constitutive-expression vector pKK233-2. The recombinant plasmid pKK233-2-SZZ-hrpN was finally transformed into P. agglomerans 308R by electroporation. The biocontrol bacterium 308R (pKK233-2-SZZ-hrpN) expressed ZZ::Harpin protein without the induction with IPTG, secreted the active protein out of the cell membrane. But the level of constitutive expression was much lower than that of the induced expression. Further work needs to be done to assess the effects of this genetically engineered bacterium for inducing plant resistance todiseases.The genetic stability of the recombinant plasmid in host bacteria is one of the important factors that affects the biocontrol effect in the field. We found that after cultivating both in antibiotic-free LB medium and on the surface of plant leaves the genetic stability of recombinant plasmid in biocontrol bacterium 308R (pKK233-2-SZZ-hrpN) were a little higher than 308R ( pCPP430) , but these two strains were all much lower than 308R ( pRTnp) carried the parDE segment which is responsible for plasmid partition. The genetic stability of 308R (pKK233-2-SZZ- hrpN) is needed to improve in order to establish the foundation for large scale fermentation and long period and stable effect in the fields without the selection pressure of antibiotic markers.