Cloning And Characterization Analysis Of Xylanase Genes From Fusarium Graminearum And Preliminary Screening Of The Target Proteins Of Wheat

Fusarium head blight or scab caused by Fusarium graminearum (sexual stage: Gibberella zeae) is an economically important disease on small grain cereal crops, prevalently in the areas with warm temperature and high humidity worldwide, In recent years, the disease is often outbreak in the yellow river-huai river area and Northwest area of wheat production, resulting in great losses.FHB which not only can cause yield loss,but also produce a variety of mycotoxins.such toxins can greatly Inhibit eukaryotic in protein biosynthesis of vivo and damage the immune systems of humans and animals.lt is very significant to understand the molecular mechanisms of the interactions between wheat and F.graminearum and to isolate resistance related genes, which should be helpful to protect plant from infection.Fusarium Graminearum in the process of infecting the host to secrete a variety of cell wall degrading enzymes such as cellulase, xylanase and pectin enzymes.beause of hemicellulose is the main component of pant cell walls,while the xylan is the component of hemicellulose,implied xylanse played an important role in spikelets organized by The ministry of pathological changes. Fusarium PH-1has been sequenced and published. We accordingly the sequence which have published design primers to clone the12xylanase genes from Fusarium graminearum.we analyzed the expression of12xylanse genes under Induced and non-induced by host. In this study, using xylanases as bait, we screened the wheat yeast expression library (constructed with wheat cultivars Sumai3) by yeast two-hybrid (Y2H). The main results are as follows:1.12xylanase genes was cloned from Fusarium graminearum which have inoculated wheat.and analysised expression though semi-quantitative RT-PCR under Induced and non-induced.we found the expression of12xylanase genes is very different. 2.12xylanase were cloned into pGBKT7vector to generate the bait vector pGBKT7-xy/anase. Activation and toxicity tests proved that they could be used as baits for screening of cDNA library except00184BKT7.3. With pGBKT7-xylanase which non-transcribed activation as baits, we screened Sumai3wheat yeast expression library which have established. five proteins which can interact with xylanse were screened out.includ Calcium lipid binding protein, translation elongation factor, stress response proteins, subtilisin-like protease, and vacuolar ATPase subunit G. the major functional regions including signal transduction,plant translation regulation, apoptosis andStress defense etc. Follow-up work may use cloning technology to obtain these proteins’ full-length sequence, and use site-directed mutagenesis or other methods to clarify the key interaction sites or amino acids.4. Interactions of FGL1and Triticum aestivumLinn iliimumphilin(named TaFKBP12) protein have been proved by Y2H,Also proved in plant cells by BIFC.TaFKBP12is intracellular receptor of the immunosuppressive drugs such as FK506in animals and humans.However, FKBP12of most plants can not combine with FK506.In this study,we found FKBP12interaction with FGL1is not disrupted by FK506in yeast cell.Thus,plant FKBP12gene is different from their mammalian.The exact function of wheat FKBP12protein should be further analyzed by RNAi or virus-induced silence.