Study On The Function Of Virulence Protein Gene AlPep1 Of Ornamental Tobacco Brown Spot Disease

Tobacco is a genus of family Solanaceae,an annual or limited perennial herb whose mature leaves are mainly used for cigarette production and have extremely high economic value.The origin of tobacco is in South America and it is widely cultivated in China now.After years of breeding,many cultivars were released.Among them,'Honghuadajinyuan' has the characteristics of wide adaptability and high output quality.It's blade is thick,inflorescence is concentrated,corolla is dark red,flowering period is longer,and its appearance is more ornamental than other tobacco varieties.So it can be used as an ornamental tobacco.Tobacco brown spot disease(TBSD)is a kind of fungal disease caused by Alternaria longipes infection in the mature stage of tobacco which seriously restricts China's tobacco production and causes huge economic losses.However,'Honghuadajinyuan' is less resistant to TBSD and has a strong suscepti-bility.After the leaves are infected by pathogens,it is easy to form large-area lesions,which seriously reduces economic value and overall ornamental value.In previous studies on the pathogenesis of TBSD,the pathogenicity of AlPepl gene deletion strain was reduced.In order to further explore the role of AlPepl gene in the pathogenic mechanism of A.longipes,this study used this gene as an entry point to provide a theoretical basis for the study of pathogenic mechanism and the development of specific antimicrobial agents of TBSD.The main results of this study are as follows:1.By analyzing the transcriptome data of A.longipes wild type strain C-00 and histidine kinase AIHK1 gene deletion strain HK?4,it was found that the relative expression of AlPep1 gene in HKA4 was higher than that of wild type strain,suggesting that the presence of AIHK1 gene could inhibit the expression of AlPep1 gene,and that AlPep1 gene was Akey factor in TCSTS.2.Through bioinformatics analysis of the amino acid sequence of AlPep1 gene,it was found that the expression product aspartic protease is most likely to be secreted extracellular protein with good stability.3.Agrobacterium-mediated Ti plasmid transformation was used to replenish the previously acquired AlPep1 gene deletion strains ?Pep1-3 and ?Pep1-5,and the complement vector was successfully constructed,waiting for transformation of fungi.4.Agrobacterium-mediated Ti plasmid transformation was used to study the subcellular localization of AlPep1 gene in wild-type strain C-00,and the localization vector was successfully constructed,and 10 localized strains were obtained,of which 2-1 and 2-9 were under fluorescence microscope.The fluorescence effect is strong,and it can be observed that the fluorescence covers the whole hyphae,and it can be confirmed that the aspartic protease of the AlPepl gene expression product is indeed distributed on the cell wall.