Functional Analysis Of Tri8 Involved In Fusarium Toxin Synthesis

Fusarium head blight(FHB)is a global plant disease,which has a great impact on wheat yield.Scab can also cause food safety risks,because the pathogenic Fusarium can synthesize deoxynivalenol(DON)and its derivatives 3-Ac DON,15-Ac DON and other toxins.Tir8 is one of the key genes involved in Fusarium toxin synthesis.Different types of Tir8 encode different proteins,which can catalyze the deacetylation of different sites on the precursor molecules,thus producing different types of DON toxins.For example,the Tri8 protein encoded by Fusarium asiatica Fa1312 can catalyze the deacetylation of intermediate 3,15-di Ac DON at 15 th carbon atoms to form 3-Ac DON,while the Tri8 protein of Fusarium graminearum Fg1230 can catalyze the deacetylation of intermediate at 3th carbon atoms to form 15-Ac DON.Expression and purification of Tri8 protein,analysis of its structure and catalytic characteristics will be helpful to clarify its mechanism of action,and to prevent and control wheat scab and the food toxin pollution caused by it.The prokaryotic expression and purification system of Tri8 protein was established.Tri8 gene was amplified by PCR and prokaryotic expression vector was constructed,which was introduced into ER 2566 to induce expression.The fusion protein was labeled with MBP and His tag.A large number of fusion proteins were purified by affinity chromatography on amylose column,then the fusion tags were cleft by protease,and the tags were removed by Ni His trap affinity chromatography column.Finally,a small amount of Tri8 protein without affinity tags was obtained.This laid a foundation for the further study of the catalytic properties and structure of Tri8 in vitro,and also provided a reference for the expression and purification of other secretory lipase family proteins.In this study,Tri8 knockout strain was obtained,and Tri8 substrate 3,15-di Ac DON was accumulated in this strain.Tri8 knock-out vector was constructed and introduced into wild-type Fusarium graminearum strain Fg p H-1 by Agrobacterium transformation.Knock-out strain was screened by resistance and PCR.Wild type strains and Tri8 knockout strains were induced and cultured,and their toxin types were analyzed by GC-MS.The results showed that the toxin chemotypes of wild-type strain was 15-Ac DON,but the precursor 3,15-di Ac DON of 15-Ac DON accumulated in the knockout strain.The results confirmed that the function of Tri8 protein was to catalyze the deacetylation of 3,15-di Ac DON,and the strain could be used for the subsequent preparation of 3,15-di Ac DON,as well as the systematic study of the catalytic properties of Tri8 protein in vitro.In this study,a Fusarium strain containing two Tri8 genes was constructed,which can be used for the preparation of DON toxin.The Tri8 encoding product of Fusarium graminearum Fg p H-1 catalyzes the deacetylation of 3,15-di Ac DON intermediate to form 15-Ac DON,and the Tri8 protein of Fusarium asiatica Fa1312 catalyzes the deacetylation of 15 th carbon atoms to form 3-Ac DON.The Tri8 from Fg p H-1 strain was cloned,and the over-expression vector was constructed.Then it was transformed into Fusarium asiaticum Fa1312 by Agrobacterium mediated genetic transformation.Finally,the target strain was identified by resistance screening and PCR.Using this strain,we hope to prepare DON toxin and avoid the interference of 15-Ac DON and 3-Ac DON.The above research laid a foundation for the further study of Tri8 catalytic mechanism and the detection of DON toxin.