| The mulberry popcorn disease is one of the most serious fungal diseases that harmed mulberry.The pathogen specifically infects the mulberry flower organ in flowering period.Mulberry shrunken popcorn disease is one kind of mulberry popcorn disease,caused by Scleromitrula shiraiana.In the study,apothecium、sickness fruit、sclerotium were collected from incidence area of mulberry popcorn disease,S.shiraiana was isolated from apothecium and DHN melanin was identified in black material produced during the infection process.DHN melanin not only plays an invaluable role in the stress environment,but also closely relates with the pathogenicity of pathogens.The biosynthesis of DHN melanin is controlled by multiple genes,and the deletion of any one gene will hinder the biosynthesis of DHN melanin affecting pathogenicity.Scytalone dehydratese is a key enzyme in DHN melanin synthesis pathway.Therefor,in the present study,the genemonic DNA and cDNA of the scytalone dehydratese were obtained by PCR using degenerated primer sets and by RACE-PCR.The knockout vector was constructed,confirming the importance of the ShSCD gene in the melanin biosynthetic pathway.The main experimental results are as follows: 1.Cloning and structure analysis of ShSCD geneDegenerated PCR primers were designed based on the consensus sequences from other fungi and the completed genomic sequences of ShSCD were then obtained with the full length of 1083 bp by the method of RACE in S.shiraiana.This gene has two introns and three exons.It was found that ShSCD had the conserver binding and catalytic residues.Sequences of ShSCD showed high similarity with those SCDs from Botryotinia cinerea T4 and Sclerotinia sclerotiorum.The secondary structure of ShSCD protein is mainly composed of alpha helix and random coil,it has a crystal structure,similar to the SCD protein in Magnaporthe grisea.2.ShSCD gene-deletion mutantsTwo fragments,fragment I(870bp)and fragment II(866bp)were obtained using genome walking technique.The ShSCD gene-disruption vector PSKH-ShSCD was constructed using PSKH plasmid and antibiotic hygromycin B.The mutants were screened by antibiotic hygromycin B.As a result,seven transformants were obtained.The PCR results revealed that the ShSCD gene was not amplified in the transformants,but the partial knockout vector sequence was amplified.Semiquantitative PCR and qRT-PCR showed that the ShSCD gene was not expressed in the transformants.Southern blotting results also indicated that ShSCD was a single copy gene in wild type and the hygromycin gene fragment,not ShSCD gene,was detected in transformants.The result indicated that the selected mutant was a positive mutant.3.Functional analysis of ShSCD geneAfter being cultured for 4-5 days,wide type colonies were gray-white in face and dark green in back.However,the ΔShSCD mutant was light yellow in face and the black was brown.The other photypes of mutant were similar to the wild-type strain.Compared to wide type strain,the mutant ΔShSCD had slower vegetative growth rate.By determining the content of melanin,the content of melanin in the ΔShSCD mutant was significantly lower than that of the wild type strain.The growth of ΔShSCD mutant was similar to that of the untreated wild-type strain.Which was inhibited when the concentration was up to 0.5M sodium chloride.In addition,the growth rate of ΔShSCD mutant was inhibited in 0.9M sodium chloride.After the treatment with H2O2(5mM),the inhibitory rate was no significant difference between the wild type strain and the ΔShSCD mutant.At a concentration of 10 mM,the inhibition rate of wild type strain was 89%,the growth of ΔShSCD mutant strain was completely inhibited.The results indicated that ShSCD gene might play an important role in mycelial growth,melanin synthesis,osmotic regulation and antioxidant capacity of S.shiraiana,which provided a theoretical basis for further study on the pathogenic mechanism of mulberry sclerotinia sclerotiorum,which provides a new idea for the development of new fungicides. |
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