Digital Expression Profile Analysis Of V. Dahliae Microsclerotium Germination And Genes Study Related With Germination

Verticillium dahliae Kleb. is a widely distributed soilborne pathogen causing vascularwilt diseases on more than660plant species in38families. It cause serious damage to cottonin cotton growing areas, becoming one of the most impotant factors in the cotton production,in the worldwide. It can survive as microsclerotia, the main initial inoculum of V. dahliae, formore than20years in soil. The aim of this study is to indentify genes related withmicrosclerotium germination and clear the molecular regular mechnism by constructingdigital expression profile of V. dahliae microsclerotium germination. Therefore, it is hope tocontrol Cotton Verticillium Wilt by controlling the main inoculum-microsclerotia. This researchused V. dahliae as material, used Solexa/Illumina digital gene expression (DGE) technology to analyze thegenes related with microscleortia germination. The target is to study the mechanism of microscleortiagermination. The main results of this research are as follows:1. Stable microsclerotium RNA extraction method was established. Sample of microsclerotia driedby Vacuum freer for12h was grinded in liquid nitrogen, then RNA is extracted by Trizol Reagent. Highquality of total RNA from microsclerotia was extracted by this modified method.2. cDNA libraries derived from germinated-and non-germinated-microsclerotia were constructed.3,671,123and3,733,211tags were obtained from VDMG-b and VDM libraries by Solexa sequence,respectively. After removal of useless tags,3,495,080and3,316,376clean tags were obtained,respectively. The “quantity” and “category” distribution of clean tags with the copy number of cleantags were statisticsed. The results showed: When the copy number of clean tags was added, the quantity ofclean tags increased, reaching a maximum when the copy number was>100; however, the categorycorrelated negatively with the copy number.3. The sequences were aligned under BlastN program using the following databases as references:NCBI database (NCBI.[http://blast.ncbi.nlm.nih.gov/Blast.cgi].) and V. dahliae genome database(http://www.broadinstitute.org/annotation/genome/verticillium_dahliae/MultiHome.html), the clean tags oftwo libraries VDMG-b and VDM were matched with the reference genes. Using these criteria,27.81%(VDMG-b)and23.25%(VDM) for “quantity” and18.87%(VDMG-b)and7.17%(VDM)for“category” were separately obtained. 4. Based on the analysis of the differentially expressed genes, we identified813up-regulated genesand470down-regulated genes.3up-regulated genes (H(+)/hexose cotransporter VDAG-07083,Fungal Zn(2)-Cys(6) binuclear cluster domain VDAG-05553and cyclopentanone1,2-monooxygenase VDAG-3493) and3down-regulated genes (FAD bindingdomain-containing protein VDAG-07578, chitin deacetylase VDAG-03333, WSCdomain-containing protein VDAG-03711) were selected for RT-PCR according to their highlyexpressed levels and potential roles in microsclerotia. Tubulin beta chain was chosen as the internalreference gene. The results showed the result of RT-PCR can reflect the result of DGE...