Part:BBa_K4832003

P.chlororaphis KC-P

Usage and Biology

In prokaryotes,RNase III is the main degrading enzyme of dsRNA. In order to increase the accumulation of dsRNA expression in Pseudomonas chlororaphis we knocked out RNase III gene of Pseudomonas chlororaphis and obtained mutant P.chlororaphis KC-P can greatly increase the accumulation of dsRNA.

Characterization

We transferred plasmids capable of expressing dsRNA into Pseudomonas chlororaphis that knocked out RNase III gene and Pseudomonas chlororaphis that did not knock out RNase III gene. Then, we conducted Northern Blot experiments to verify whether the accumulation of dsRNA increased after knocking out RNase III.

Figure 1. Northern BlotThe first lane is the RNA of P. chlororaphis KC-P, which is transferred into the plasmid that can express dsRNA; the second lane is the control designed for the first lane; the third lane is the RNA of wild Pseudomonas chlororaphis, which has not been transferred into the plasmid . The fourth lane is RNA from wild Pseudomonas chlororaphis that has been transferred into a plasmid that can express dsRNA, and the fifth lane is a control designed for the fourth lane.

From the results of Northern Blot, it is obvious that we knocked out the RNase III of Pseudomonas chlororaphis ,which greatly increased the accumulation of dsRNA.

References

[1]  Kupferschmied, P., Maurhofer, M. & Keel, C. Promise for plant pest control: root-associated pseudomonads with insecticidal activities.Front. Plant Sci.4, 287 (2013).

[2]  Keel, C. A look into the toolbox of multi-talents: insect pathogenicity determinants of plant-beneficial pseudomonads.Environ Microbiol18, 3207-3209 (2016).

[3]  Péchy-Tarr, M.et al.Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains ofPseudomonasfluorescens.Environ Microbiol10, 2368-86 (2008).

[4]  Schellenberger, U.et al.A selective insecticidal protein fromPseudomonasfor controlling corn rootworms.Science354, 634-637 (2016).

[5]  Anderson, J.A.et al.Agronomic and compositional assessment of genetically modified DP23211 maize for corn rootworm control.GM Crops Food11, 206-214 (2020).

[6]  Wei, J.Z.et al.A selective insecticidal protein fromPseudomonasmosselii for corn rootworm control.Plant Biotechnol.J. 16, 649-659 (2018).