Studies On Fungal Chitin Synthases And Its Inhibitors

Chitin,which is synthesized by chitin synthase,is one of the most important component of fungi cell wall,but absent from plant and mammalian species.There are 7 classes of chitin synthase isoenzymes presented in the fungal kingdom.The yeast-like fungi usually have chitin synthases of classⅠ,ⅡandⅣ,the last two of Which play a key role in yeast survive and infection.While the filamentous fungi have 7 classes of the chitin synthases,and the important classses areⅢ,ⅤandⅥ.It is important to study fungal inhibitors for fungal disease control. This study is aimed to screen for inhibitors from soil microorganism and Chinese traditional medicine.The screen were done by different method using the Saccharomyces cerevisiae YFK32 as indicator,several strains having inhibitory effect were obtained by 96-wells method from soil, while several Chinese traditional medicine were found via filter paper method. Calcofluor white can inhibit the growth of Saccharomyces cerevisiae, while the inhibition of yeast growth can be rescued by chitin synthase inhibitors.In this study,the chitin synthases were selected as target of inhibitor.The positive screening method was done by applying the sample to the plate,which had YFK32 as indicative strain presented with inhibitory concentration of calcofluor white.Several putative chitin synthase inhibitors were found by this method from Chinese traditional medicine extraction,but none of soil microorganism was found having such activity so far.For the reason that different inhibitors target different chitin synthase isoenzyme,and the defective strain which has only one chitin isoenzyme increased susceptive with target inhibitor while comparing with wide type. S.cerevisiae,which lack of chs1 and chs3,containing only one isoznzyme CS2 was constructed to further comfirm the precise mechanism of putative inhibitor.But none of the inhibitors was found as CS2 specific inhibitor yet,the precise target were under researching.Furthermore,to better comprehend the function of filamentous chitin synthase,the chs V of Botrytis cinerea was studied.A 1.5 kb upstream flanking and 1.3 kb downstream sequence of chs V gene were amplified by PCR.The fragments were cloned into MCS of pAG32 located the flanking of HygB,separately,to form gene disruption cassette.The gene disruption cassette was then subcloned into binary vector.After transforming the binary vector containing chs V gene disruption cassette into the Agrobacterium tumefaciens EHA105,the strain was used for gene disruption to gain better transformation efficient.