| Sclerotinia sclerotiorum which parasites oil crops and cruciferae belongs toascomycetes. It could do a great harm to our conutry gain production when it wasinvaded into the host. Sclerotinia sclerotiorum is a kind of facultative andhomothallism hyperparasite. Ascospores produced from sexual reproduction is themain factor of Sclerotinia. Therefore, studying the function of MAT-2gene related tosexual reproduction can reveal the molecular mechanisms of sexual reproduction ofSclerotinia sclerotiorum and open up a new way to prevention.1In this study, full length cDNA of MAT-2was cloned from the total RNA ofmycelium of Sclerotinia sclerotiorum. After sequence analysis, it is found that thegene contains the conserved domains of HMG-box, the domain encoding a DNAbinding protein. It has an open reading frame of1185bp, encoding394amino acidresidues with a deduced molecular weight of44.73kD, which pI is8.13. Bybioinformatics analysis, we determined the positional relationship between MAT-1gene and MAT-2gene and we found that the MAT-2gene and MAT-1gene exists inthe same chromosome of Sclerotinia sclerotiorum genome, and the spacing of thetwo genes is1657bp. Phylogenetic tree was constructed, MAT-2gene was obtainedin the first branch, and a higher homology with the MAT-2gene of other strains. Byprotein secondary structure prediction, it is found that the protein encoded by MAT-2contains48.48%random coil. We also Successfully constructed the prokaryoticexpression vector and transferred into E.coli BL21. SDS-PAGE showed that MAT-2gene was expressed as a48.2kD fusion protein when induced with IPTG. Thisexperiment is the foundation for the preparation of specific antiserum.2In this study, expression dynamics of the MAT-2gene was detected with themethod of Real Time PCR. In this experiment, we use mycelium, different periods ofsclerotia and apothecia as materials. Total RNA of Sclerotinia sclerotiorum wasextraction for the Real Time PCR reaction. Identified the specificity of the primers through the solubility curve can be used for the Real Time PCR detection. Weproform the Real Time PCR detection by the method of relative quantitative PCR.We also set up two housekeeping gene as references. It is found that MAT-2geneexpression in the mycelium is the lowest. The expression of the period of sclerotia5,6was significantly higher than other periods. The expression of5period of sclerotiais more than6periods may be due to the internal mycelium of6period sclerotiadecreased activity, therefore, the MAT-2gene expression was slightly lower than theperiod5. Using quantitative PCR to detect the temporal dynamics expression of theMAT-2gene, which enriching the sexual reproduction of Sclerotinia sclerotiorum.3In this study, we have prepared the protoplasts by using Glucanex enzymatichypha of Sclerotinia sclerotiorum and successfully construct the knockout fragmentsby using overlapping PCR. We transformed the knockout fragments into protoplastsby the method of PEG-CaCl2. After protoplasts regenerated culturation andhygromycin resistance screened culturation, we have gained the knockout MAT-2mutant transformants and analysised the biological characteristics and pathogenicity.In this study, using a variety of PCR amplification to verify the knockouttransformants. Compared with wild-type strain, it is found that the knockouttransformants have some changes in mycelium dense extent, the number of sclerotiaformation and the angle and the number of hyphal branch. Compare the wide type ofSclerotinia sclerotiorum with the knockout transformants, it is found that pathogenicof knockout MAT-2gene transformants have significantly lower than the bypathogenicity of wild type strain. This experiment determined the relationshipbetween mating genes and pathogenicity, which laid the foundation for themechanism of sexual reproduction of Sclerotinia sclerotiorum. |
PDF Full Text Request