Construction And Analysis Of Random Insertion Mutant Library Of Fusarium Graminearum

Fusarium head blight (FHB) caused by Fusarium graminearum is a serious disease of cereal crop, distributed widely in warm and humid areas all over the world, endangering a variety of cereal crops, like wheat, barley, oats, corn, rice, rye, etc.. FHB not only lead to crop production cut, and also produce doexynivalneol, zearalenone and other toxins, can cause vomiting, abdominal pain, dizziness and other symptoms of poisoning after eating, threat to human and animal health seriously. As complete genome sequencing of F. graminearum finished, researchs on gene function and pathogenesis mechanism became an effective way to scab control. The isolation and identification of F. graminearum genes involving growth, development and pathogenicity can be accelerated by the establishment of a random insertion mutant library.Restriction enzyme mediated integration (REMI) was firstly developed by Schiestl and Petes in Saccharomyces cerevisiae. In recent years, REMI used widely in mutant library establishment, gene tagging, etc. for its high transforment efficiency, high percentage of single-copy insertion and its easiness to separate mutant genes. Currently, this technology has been applied widely in yeast, filamentous fungi and animal studies.In this study, we had explored and optimized the conditions of protoplast preparation, purification, and restriction enzyme mediated transformation, and then established an effective REMI transformation system. After several experiments, we obtained more than 2300 hygromycin B resistant transformants and constructed the mutant library, and furtherly carried PCR, Southern analyzing and phenotype, virulence screening of some transformants.After optimization, the maximum efficiency is 68 transformants each transformation, two to three times to normal protoplast transformation; PCR amplification of 15 transformants showed that they all contain the hygromycin B resistance gene; The result of Southern blot analysis of some transformants indicated that the percentage of single-copy insertion is 66.7%,23.9% higher than normal protoplast transformation.