A Study On Effect Genes Of Ice Nucleoprotein InaQ Of Pseudomonas Syringae

Pseudomonas syringae is a common plant pathogen that can cause a variety of plants to produce hypersensitivity, and it’s also a strain of INA bacteria that can induce plants frost damage at higher temperatures. The key factor in inducing freezing injury is a secretion protein-ice nucleation protein(INP). In this study, we study on the Pseudomonas syringae MB03which has a high ice nucleation activity. We construct a transposon mutant library of MB03, and study on the effect genes of the ice nucleation activity.Pseudomonas syringae MB03has a high ice nucleation activity and its INP gene inaQ has been sequenced. In this study, we built a transposon mutant library of MB03using the suicide transposon pUT-mini Tn5-Km2by the method of two parents joint.We analyse the INP secretion by immunological method with the antibody of inaQ. We initially screened the mutants with in site hybridization and get the mutants with significant changed INP secretion. Then these mutants are confirmed by Cy5flow cytometry.An improved TAIL-PCR method is used to identify the insertion sites using3nested special primers and some degenerate primers.The target fragments are selectively amplified and the amplified products can be directly sequenced. By this method we get the sequence flanking the insertion site from several stains with changed INP secretion activity. We compared the sequence with MB03genomic sequence,and identified some inactivated genes which is interrupted by mini-Tn5: citrate synthase gene (glt A), a glycosyltransferase gene and a2-oxo-dehydrogenase genes.Citrate synthase gene (glt A) was choosed to be conducted a expression compensation.The inaQ expression of the wild-type stains and the compensation stains are compared by Real time-qPCR and Western blot. It’s confirmed that gltA interruption can lead to reduced inaQ expression.