Screening And Function Research In The Pathogenic Genes Of Alternaria Longipes Regulated By Histidine Kinase AlHK1

Tobacco brown spot disease is occurred in the mature period of tobacco caused by Alternaria fungal.Tobacco brown spot disease restrict Chinnese tobacco cultivation seriously.It bring about huge economic losses.In recent years,the pathogenic mechanism of Alternaria alternata has attracted people’s attention.In order to explore the pathogenic mechanism,we start from the relationship between signal transduction and pathogenicity.By suppression subtractive hybridization and RNA-seq,get pathogenicity related genes which regulated by AlHK1.To identify the genes involved in pathogenicity,a suppressive subtractive hybridization pproach was applied to reveal profiling of differentially expressed genes.About 21 unigenes were identified from forward SSH library which involved in pathogenicity.Study with the aim genes by gene knockdown.Provide a theoretical basis in its specific antimicrobial agent:1)By means of SSH,two subtracted cDNA libraries were constructed which involved in AlHK1.Then,obtain a total of 211 positive clones.In the end,we selected 21 genes which involved in pathogenic,signal transduction,metabolism function.2)Using homologous recombination method,knocking out 9 candidate genes.Through the protoplast transformation,hygromycin B selection,and success of obtaining 3 gene knockout transformants in the end.Fortunately,these transformants are in differ from the wild type more or less.3)By laser scanning is confocal microscope to C-00 and HKΔ4 infection were observed,establishment the RNA-seq library involved in different infection time points;Using Illumina HiSeqTM 2000 sequencing platform and successfully construct the in different infection stages of gene expression library;Application of biological information science and technology to analyze the different library of gene expression.The library contain a large number genes which are expressed differently.