Targeting Vectors Construction Of The Mating Type Genes MAT1-1and MAT1-2and The Establishment Of Transformation System For Stemphylium Botryosum

The fungal genus Stemphylium is an important group of filamentousfungi, most species of this genus have unknown sexual state in nature,but the others are self-fertility. The research reported that someStemphylium species’ MAT loci contained a single gene, either MAT1-1orMAT1-2, whereas others contained a unique fusion of the MAT1-1and MAT1-2regions. In all fused MAT regions, MAT1-1was inverted and joined to aforward-oriented MAT1-2region. The novel structure of the loci can bewell materials for insighting into the molecular nature of sex, sexualspecification and evolution to advance our understanding of sexualreproduction.Stemphylium botryosum contains the two genes, and can be induced tosexual reproduction in laboratory. In order to continue investigationsof mating system and other related genes’ function, this reseach focusedon first step of the constuction of targeting vectors for mating type genesMAT1-1/MAT1-2and establishment of transformation system for Stemphyliumbotryosum.The standard culture of S. botryosum (CBS122441) as the material getfrom Centraalbureau voor Schimmelcultures(CBS), Utrecht, The Netherlands,and deposited in the Department of Plant Pathology, Shandong AgriculturalUniversity (HSAUP).This research gets the sequences of MAT1-1and MAT1-2according tothe messages from other papers, and use the Southeren blot technologytested the copies of the two genes, to make sure the efficient knock out.Then amplify the left and right flanking regions of the two genes, usethe vector named pBIG3C28a constuct two targeting vectors for both genes.The two vectors named pBIG3C28a-1R-1L and pBIG3C28a-2R-2L. During thesework, two transformation systems for S. botryosum are established thatfind out the perfect conditions of PEG-mediated transformation and Agrobacterium tumefacines-mediated transformation. In the PEG-mediatedtransformation system, the key point is the protoplasts preparation andregeneration, during these experiments, find that the combination oflyase (15mg/mL)/helicase (10mg/mL)/lywallzyme (10mg/mL) and celluase(15mg/mL) to digest the hyphae cultured about36h for3.5hours at30℃, and use0.7mol/L NaCl as the osmotic stabilizer can be the bestcondition for preparation of protoplasts. And use1mol/L sucrose as theosmotic stabilizer for the regeneration. After the PEG-mediatedtransformation can get many transformants. The Agrobacteriumtumefacines-mediated transformation, it will be better to use300μmol/LAS induce the Agrobacterium tumefacines （OD600=0.8）to infect spores(105/mL), co-culture at28℃for72hours.These targeting vectors and transformation systems are successful intransforming, and all of them can be the foundation of the further researchof Stemphylium.