Cloning And Functional Analysis Of Pathogenicity Related Gene VdMFS1on Verticillium Dahliae

In the current study, we measured the physiological properties of verticillium dahliae strain Vd080.In addition, we also analysed the function of pathogenicity related gene VdMFS1. Preliminarily, weconstructed the knock-out plasmid pCB1302-MFS, and successfully deleted VdMFS1gene byAgrobacterium tumifaciens Mediated Transformation (ATMT).The main conclusions are presented asfollows:1. The3isolates were identified based on temperature reaction, using V. dahliae defoliating andnon-defoliating type-specific primer (D-1/D-2, ND-1/ND-2). Results showed that V. dahliae strainsVd080and V991exhibited defoliating nature, however, Vd001was identified as non-defoliating.Thepathogenicity results also revealed significant difference for disease percentage and index. The highpercentage of diseased plants was recorded for Vd080(67.4%) and V991(67.0%) compared to Vd001(35.4%) with52.4,51.8and20.0disease index27d after inoculation (DAI), respectively. However, nosignificant differences were found between the two strains Vd080and V991.In addition, results ofstatistics defoliation rate manifested that Vd080and V991began to defoliate after15DIA, but Vd001started defoliating18DIA. Defoliation rate of Vd080and V991were significantly higher than Vd00121-29DIA.It indicates that Vd080belongs to high virulent and defoliating type strains.2. Vd080、V991and Vd001showed nucleate type colony growth on PDA medium; however, thegrowth rate of Vd080and V991were significantly higher(4.39and3.81mm d-1) than Vd001(3.38mmd-1)；In addition, the spore production of Vd080was significantly lower (37.3×107) than V991(45.9×107), however, spore production of both strains were higher than Vd001(9.2×107); The crudetoxin of Vd080(33.45μg mL-1) and V991(33.63μg mL-1) was higher than Vd001(20.00μg mL-1),however, there were no significant differences between Vd080and V991.3. According to gene annotation and blast of lettuce V.dahliae Vd17database, we get a geneVDAG09647that expressed the product, ultimately can reduce the pathogenicity, so we designed highspecific primers and cloned a gene named VdMFS1from Vd080. The length of gene was1948bpcontaining8exons and7introns.At present, it has no detail report about the function of VdMFS1incotton V.dahliae. The result of southern blot showed that there was only one copy for VdMFS1onVd080. This evidence provides a premise for researching gene function by knock it.4. Based on pCAMBIA-1302, we connected the upstream sequences of the gene UP by HPH genesequence of the selection marker and downstream sequence DOWN by end to end in the order insertedinto the vector through the digestion method. As a result, the VdMFS1gene-replacement vectorpCB1302-MFS was successfully constructed. Finally, we transferred it into Agrobacterium tumifaciensand go the targeted transformant by ATMT and antibiotic screening; the following PCR analysis provedthat the VdMFS1gene had been knocked out successfully.