Analyze Of Genes Related To Virulence Using RAPD-PCR And Study On The Function Of VdWDR5B Protein In Verticillium Dahliae

Verticillium wilt is one of the most destructive soil-borne fungal diseases,which seriously affects cotton production throughout the world.Currently,there is no effective measure available to control this disease.Hence exploring the molecular mechanism of verticiillium pathogenicity is very important for theory and practice.In addition to enhancing the host resistance,understanding the features of the pathogen and identifying key factors involved in pathogenicity will facilitate the development and deployment of effective disease control strategies.Herein,we research the Verticillium wilt from the perspective of Verticillium dahliae Kleb.Firstly,in older to identify the different genes between the strains of different virulence,RAPD method was adopted.The Y-37 primer yielded three characteristic bands,one of these at 700bp approximately,which occurred in strong and medium toxic strains only,the remaining two bands at 250bp-500bp,which occurred in weak toxic strains.Through subsequent analysis,although the three bands are not in coding regions,we can develop the three bands into RAPD markers,which will provide sequence information for establishing a normative,rapid and simple method for distinguishing strong toxic strains from weak toxic strains.Besides these,RAPD was used to identify differential expressed genes in response to cotton root.Three candidate primers were selected for RAPD analysis after screening 40 random primers for their discriminatory abilities.Y-1,Y-40 primers produced down-regulated bands;Y-21 produced up-regulated bands.After sequencing,the bands were identified by blast search of the VdLs.17 whole genome database.The blast records indicated that the fragment yielded by Y-1 was a part of heat shock protein,the fragment yielded by Y-40 was a part of high-affinity glucose transporter SNF3,the fragment yielded by Y-21 was a part of WD repeat-containing protein 5B(WDR5B,VDAG 06686).Finally,we studied the function of VdWDR5B that was up-regulated induced by cotton root using gene knockout method.At the morphological characteristics aspect,gene deletion mutant displayed a more elongated shape in conidium,abnormalities in spore germination,frequently and irregular branching in hyphae growth,losing the ability to form microsclerotia,a changed vacuolar morphology,drasticly decreased conidiation in the liquid culture medium,slower growth pattern on agar media compared to the corresponding wild type.At the pathological characteristics aspect,gene deletion mutant has the following characteristics compared to WT:the ability of adhesive on root was not affected,but the penetration was impaired,moreover,in the case of root damage,the virulence of mutant was enhanced,while in the case of root unwounded,the virulence of mutant was weakened.