Functional Analysis Of Endo-1.4-β-xylanase Gene Ss-Xyl1 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum（Lib.） is an important pathogenic fungi. It has a wide host range and can infect many crops. The rape sclerotinia rot is caused by Sclerotinia sclerotiorum and can bring huge economic losses not only to our country but also to the world’s agricultural production. So far, the molecular mechanism of pathogenicity and the growth of Sclerotinia sclerotiorum are still unclear. The preliminary studies found a hypothetical xylanolyt ic hydrolase gene Ss-Xyl1 in Sclerotinia sclerotiorum, analyzed the basic characteristics of the gene and its function in the process of pathogenicity. The result can provide new clues for revealing the mechanisms of pathogenic and growth development of Sclerotinia sclerotiorum.1. The basic features study of Ss-Xyl1 gene in Sclerotinia sclerotiorum: Analysis ing the protein encoded by Ss-Xyl1 find it has an n-terminal signal pept ide structure of 21 amino acids, and the c-terminal has a Glyco_hydro₁1 structure. The protein of Ss-Xyl1 is highly similar with the endo-1.4-β- xylanase in many fungi. Ss-Xyl1 protein is similar to the BC1G₁3645 protein from Botrytis cinerea and the sim ilar ity reach 91%（E value of 3e-143）. Ss-Xyl1 was transferred to Pichia expression to acquire the active protein. And DNS method was used to measure the xylanase activity. The results show Ss-Xyl1 transfer Pichia the xylanase activity was 9.8845 U while the xylanase activity of the control strain w as 0.1068 U, that shows Ss-Xyl1 has xylanase activity. During the sclerotia carpogenic germination process and the sclerotia developm ent, Ss-Xyl1 has a high expression but the highest of the Ss-Xyl1 is in the sclerotia germinat ion process, and the expression amount is three times than the mycelium stage. The Ss-Xyl1 gene expression quantity rise in the early infection of Arabidopsis thaliana significant ly. The highest relative expression of Ss-Xyl1 is the xylan as the carbon source, it has the same trend to other strains when xylan was used. This indicating that the Ss-Xyl1 gene expression in Sclerotinia sclerotiorum is induced by the xylan.2. The function of Ss-Xyl1 gene: Using “Split-marker” strategy knockout the Ss-Xyl1 in Sclerotinia sclerotiorum, and adopt the principle of complementarity ectopic make the gene up. The colony of the deletion mutant strains KO52 mycelial growth rate slowly than before, and the tip of the hyphae branches densely. The pathogenicity in vitro rape leaf and arabidopsis plants is signif icantly reduced in the deletion mutant strains KO52. The mycelial growth rate, the tip of hyphae and the pathogenicity of complementary transformant KOC5 are similar with the wild type strain. The result indicating that the gene may be closely related to the mycelial growth and the pathogenicity in Sclerotinia sclerotiorum. Using DNS method measuring the xylanase enzyme activity showed that KO52 xylanase activity only 0.13 U, obvious ly lower than the wild type strain 1980 which xylanase activity was 0.37 U. The sclerotia half-buried in sand which was sterilized to observed the sclerotia carpogenic germination. The sclerotia produced by KO52 strain found not germinate with the formation of carpogenic germination and not germination ascus handle. This described that Ss-Xyl1 is related to the sclerotia carpogenic germination in Sclerotinia sclerotiorum.In summary, Ss-Xyl1, an endo-1.4-β- xylanase gene in Sclerotinia sclerotiorum is associated with the mycelial growth, the pathogenicity, and the sclerotia carpogenic germination. In-depth study of the gene can provide a new clues, to prevention and treatment of sclerotinia.