Functional Study Of Gene SsAna In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum parasitizes a variety of plants,causing S.sclerotiorum stem rot and other diseases.S.sclerotiorum usually leads to plant necrosis and causes great damage to crops when S.sclerotiorum infects plants.In China,S.sclerotiorum occurs in rape planting areas.Therefore,it is of great significance to study the pathogenic mechanism of S.sclerotiorum and find a way to solve the disease caused by S.sclerotiorum.?-L-Arabinofuranosidase exists widely in fungi and is involved in hemicellulose hydrolysis together with other hydrolase,which is closely related to the integrity of cell wall.Studies have shown that?-L-Arabinofuranosidase is associated with fruit softening.Anabfa has also been extensively studied and expressed in some fungi,such as aspergillus niger and neptuni.However,the effect of?-L-Arabinofuranosidase has not been reported in S.sclerotiorum.In this experiment,the seed sequence of?-L-Arabinofuranosidase from aspergillus nidulans was searched in the genome of S.sclerotiorum,and a highly homologous sequence sscle?04g034810 was obtained,And this gene was named SsAna,and the complete sequence of the gene SsAna encoding?-L-Arabinofuranosidase was cloned from the genome of S.sclerotiorum,the prokaryotic expression vector p GEX-Ana and transient expression vector PCNFe GFP-Ana were constructed,and the knockout vector RH-SsAna was obtained by gene fusion,then,PEG-mediated protoplast transformation was used to obtain the quasi-transformants,two homozygous transformants A1 and A2 were obtained.The function of SsAna in S.sclerotiorum was observed,the main results were as follows:(1)The Mutant A1 and A2 lost the ability to produce sclerotiorum,and the growth rate of Mutant A1 and A2 was significantly lower than that of wild type 1980.(2)The Mutant A1 and A2 were sensitive to H₂O₂ and Congo Red.(3)The pathogenicity of the Mutant A1 and A2 was significantly smaller than that of the wild type 1980 in tobacco,Tomato and rape leaves in vitro.(4)The mycelial growth branches of knock-out mutants A1 and A2 were abnormal.(5)Transient expression of tobacco soakings and analysis of biological information of proteins.It was found that the protein was a secretory protein and located on the cell membrane.(6)After prokaryotic expression,the protein was found to be unstable and the enzyme activity was highest at 65?for 30 min.The results indicated that SsAna gene may be involved in the formation of sclerotiorum and affect the growth rate and pathogenicity of S.sclerotiorum.