The Preliminary Study On The Function Of The Polygalactose Acid Enzyme Gene In Valsa Pyri

Pear Valsa canker is an important stem diseases in Northern areas of pear, In severe northern pear pear incidence areas often cause a lot of dead trees or ruin garden, after the pear was infected by bacteria, the cell wall was degradated and showing bark rot symptoms. However, the research about pectinase gene has not been reported. We analysized the transcriptome data of the strong virulent strain F-HN-6 and weak virulent strain F-HN-2a-1, and an upregulated polygalacturonic acid enzyme gene Vamepg was predicted finally. The function of Vamepg gene in the process of pathogen infection was explored by knockout method, the results were as follows:1, The expression levels of Vamepg gene was detected using qRT-PCR, results showed that the strong virulent strain is 1.86 times higher than weak virulent strain in Vamepg gene expression levels. Analysis predicted that the Vamepg gene encode exopolygalacturonic acid enzymes with exocrine function, the enzymes can be secreted outside the cell membrane2, Linearized vector was constructed by Double-joint PCR. Mutants of the wild strain that can grow normally on medium containing hygromycin were selected through the PEG-mediated protoplast transformation technique. Then single mutants were confirmed by PCR with four pairs of primers and Southern Blot, and found four deletion mutants of Vamepg. With the help of the gene knockout technique, two complemented mutants of Vamepg were constructed. The gene of Valsa pyri can be knockouted through the PEG-mediated protoplast transformation technique.3, The deletion mutants of Vamepg showed that growth rate was slightly lower and pycnidia was no different in size and shape with the wild strains on PDA medium. However, the pathogenicity of the deletion mutant of Vamepg showed significant reduction, when they were inoculated to pear twigs. The complemented mutants of Vamepg gene was equal to wild-type in growth rate and pathogenicity. The enzyme activity was determined and found that the wild-type strain was growing and reached a maximum at 10 d, but the mutant strain has been maintained at a low level and not appearde maximum at the 10 d. So we speculated that deletion mutants of Vamepg gene may result in the secretion reduce of exopolygalacturonic, then reduced pathogenicity in pear twigs.