Preliminary Study Of Two Hexokinase Gene Function In Valsa Mali

Apple valsa canker, caused by Valsa mali, is one of the major diseases of appleproduction in China. In recent years, apple valsa canker has spread rapidly in every appleplantations of China, especially in the north, causing great losses each year. Traditionalmethods, such as physical control, chemical control and breeding of resistant varieties, are notoptimistic in the management of apple valsa canker. Therefore, it’s important to search for thekey pathogenetic gene of V.mali. In order to reveal the molecular pathogenic mechanism ofV.mali and provide theoretical basis and methods for disease prevention and control, the rightflanking sequences of the T-DNA insertion sites of one pathogenic mutant x1387wasamplified and analyzed. Then further preliminary study of the genes function were studied.The results are as follows:1. The flanking sequence of x1387was obtained by TAIL-PCR, and the noting resultwas Hexokinase.2. By combining the results x1387flanking sequence and genomic information, twohexokinase gene Vmgxk1and Vmhxk1were performed gene knockout. Knockout casseteswere constructed by Double-joint PCR, connecting the reporter gene with upstream anddownstream sequences. Then,64and123mutants of the two genes, Vmgxk1and Vmhxk1,were obtained by PEG-mediated transformation of protoplasts. At last,3and1deletionmutants were finally got for Vmgxk1and Vmhxk1after PCR and Southern Blot analysis.3. Experiement of phenotype between the deletion mutants and wild type03-8wereperformed systematically. The results showed that, there were no significant differencebetween the Vmgxk1deletion mutant and03-8in the growth rate, colony color of aerialmycelium and pathogenicity. But the time of producing propagules of Vmgxk1deletionmutants had delayed about15days. Campared with the wild type, Vmhxk1deletion mutantshowed slow colony growth rate, mycelium sparse, reduced pathogenicity in both leaves andbranches and generated a25days delay in the time of producing propagules, whichdemonstrated in line with x1387. All these indicated that Vmhxk1was correlate with thegrowth and pathogenicity. Both the two genes were associated with propagules producing.After verification of complement experiement, the phenotype and pathogenicity of complement mutants were similar to wild type03-8, which further proof that the defects ofphenotype and pathogenicity of the deletion mutants are due to the gene knockout.