The G Protein Alpha Subunit Mediates Sexual Developmental Processes Of Stemphylium Eturmiunum

The Stemphylium genus is an important group of filamentous ascus fungus.The heterotrimeric G protein factors, which including alpha, beta and gamm subunits, are present in many filamentous ascus fungus. Each subunit of the G protein could be encoded by multiple genes.Especially,the alpha subunit plays a vital role in mating, the identification of sex cells and the pheromone conduction of fungus. The G protein alpha subunit can also activate pheromone factors to be involved in the sexual process of fungus heterocysts via connectting the cell surface receptors with the cytoplasmic protein effectors.Most species of the Stemphylium genus are asexual in the whole life cycle, but the others are self-fertility in nature. Three types of MAT loci from Some Stemphylium species have been found:a unique fusion of the MAT1-1 or MAT 1-2; whereas others contained a unique fusion of the MAT1-1 and MAT1-2 regions. In all fused MAT regions, MAT1-1 was inverted and joined to a forward-oriented MAT1-2 region. The Stemphylium eturmiunum EGS29-099, a typical kind of Stemphylium, contains two mating genes(MAT 1-1 and MAT1-2) and can be induced to sexual reproduction on CM media at 25? with a light/dark cycle. The characteristics of sexual reproduction is producing black perithecia and ascospore.In order to study deeply the function of G-protein alpha subunit and its influence on the sexual state of Stemphylium eturmiunum, one gene-GPA1 of the G-protein alpha subunit from Stemphylium eturmiunum was knocked out and complemented through the modified Agrobacterium tumefacines-mediated transformation system in this paper. Hence this paper makes a preliminary research on the relationship between GPAI gene and sexual reproducton of S. eturmiuna.The Stemphylium eturmiunum was used as our experimental material that was kept in the fungi and fungal resource utilization laboratory of Shandong Agricultural University. The research contents are mainly included in this paper as following:accordng to the reported information of G protein alpha subunit gene of other fungi, we have designed degenerate primers and amplified the alpha subunit gene of G protein(GPA1) of Stemphylium eturmiunum; Then the flanking sequences of GPA1 gene were amplified by Tail-PCR;In addition, the knockout and complementary vectors of GPA1 gene were constructed;After the establishment of Agrobacterium tumefacines-mediated transformation system for Stemphylium eturmiunum,the knock-out mutant strains and complementary transformants were obtained; Under the same suitable condition,the wild type strain, knock-out transformant and complementary transformant were induced to produce sexual state. We find that the knock-out mutant strains cannot form mature perithecia and ascospores;the hyphae grow slower than wild type;the conidia germination decreases significantly. While the wild type strains and complementary transformants can form mature perithecia and ascospores by induction.There were no significant differences in the growth rate of hyphae and the amount of conidiophores.The construction of knockout vectors and complementary vectors of Stemphylium eturmiunum GPA1 as well as the establishment of Agrobacterium-mediated genetic transformation systems lays a good foundation for further research of Stemphylium eturmiunum other genes.