The Risk Assessment Of Foreign Genes Inserting Into Fusarium Graminearum On The Don Biosynthesis

Fusarium head blight（FHB）,caused byFusarium graminearum,is a destructive fungal disease of wheat in amost every wheat growing-area of the world.In addition to reducing seed mass and quality,the fungus contaminates grain with trichothecene mycotoxins such as DON that threatens the health of human and livestock.Genetically modified（gm）technology,as a way of genetic breeding,was extensively used in agricultural production.Now,we are faced with more and more genetically modified foods.With the further research and application of genetically modified food,its security problem was also gradually exposed.Scientists hoped to prove the safety of genetically modified food through animal feeding experiments and Changes of various kinds of ingredients in GM foods.In this article,we will use Fusarium graminearum that can produce DON mycotoxin as research material,and insert hygromycin phosphoric acid transferase gene（hph）and Bt toxic protein gene（CrylAc）into the genome of Fusarium graminearum through the way of site insertion and radom insertion Then we assess the risks of exogenous gene insertion by detecting the ability of the mutants producing DON.MBC and other benzimidazole fungicides which are used to control FBH,have caused drug resistance resistance.Studies have shown that mutation at codon 167,198 on β2-tub of field resistant strains could improve the ability of DON biosynthesis.High-throughput transcriptome sequencing of mutant at codon 167 on β2-tub showed that 1036 were up-regulated and 401 down-regulated.So,we want to know the effect of DON by foreign gene site-insertion.We used hygromycin b phosphotransferase gene（hph）and Bt toxic protein gene（CrylAc）inserting into codon 167 and 198 on β2-tub of fusarium graminearum.The result was that whether the same gene inserted into different positions or different gene inserting into the same locus,DON biosynthesis ability and tri5 gene expression level of these mutants reduced the same times.Thus,we can speculate that the risks of different foreign gene site-insertion are likely to be consistent.The random insertion of foreign gene has lots of uncertainty.In order to study the effect on DON production with the random insertion of foreign genes,we transferred hygromycin B phosphotransferase gene（hph）and Bt toxic protein gene（CrylAc）into the genome of Fusarium graminearum randomly by the method of protoplast genetic transformation.We got 1000 random insertion mutants each type in total,and then we randomly chose 250 from these mutants to analyze gene expression of tri5 by RT-PCR,respectively.Our research showed that the ratio of hph insertion mutants between up-regulation and down-regulation more than twice was about 1:1,and the ratio between up-regulation and down-regulation more than 5 flods was 1.3:1.For CrylAc insertion mutants,the ratios were 3.4:land 6.3:1,respectively.From these results,we conclude that different foreign genes have a different influence on the production of DON.CrylAc may have a little bias when it was inserted into the genome of Fusanrium graminearum.So,the method of random insertion was risky.Target DNA segments sequcences flanking hph and CrylAc sequence of the random insertion mutants whose expressions of tri5 gene were more than twice up and down-regulation by tail-PCR and blast in Genebank offusarium graminearum.We totally got 29 genes,including 10 down-regulated genes and 7 up-regulated genes from hph insertion mutants,6 down-regulated genes and 6 up-regulated genes from CrylAc insertion mutants.Now,we chose FGSG₀1445 and FGSG₀5569 which inducd twice up-regulation and 11 folds down-regulation expression of tri5 gene when CrylAc inserted in these two genes,then we use the other two foreign genes（hph and neo）inserting in the same site of FGSG 01445 and FGSG 05569.We found that these three genes effects on the expression of tri5 and DON production were the same.The above results proved that site insertion of foreign genes to DON metabolism was safe.