Studies On Genetic Selection, Clone And Expression Of The Genes Realted To Tea Toxin Metabaolism Of Alternaria Alternata

Croftonweed (Eupatorium adenophorum Spreng.) is one of the most important invasive alien weeds in China. Chemical, ecological and biological controls have been attempted to control this weed. However, chemical control is one of the most effective but impractical in most cases. Compared to herbicides, bioherbicide has advantages in specificity, selectiveness and friendship to environments. Alternaria alternata (Fr.) Keissler was firstly isolated and recognized as a natural pathogenic filamentous fungus from the weed in our laboratory. This fungus can produce a toxin TeA, which has been identified as a photosystemⅡ inhibitor. TeA demonstrated good control effect, quick activity and broadspectrum including graminaceous weeds and broadleaf weeds. TeA has a potential to become an alternative agent for a biocontrol of croftonweed and other weeds. However, the fungus only produced the toxin at very low level through fermentation so that this production could not reach commercial level. Hence, an alternative approach is to improve toxin metabolism of the strain through biotechnology. It needs to study metabolic pathway of toxin and the related genes that influence toxin biosysthesis. The gentically modified strain would be constructed through key genes clone and transfer.An mutant 001 strain that produces little toxin was previously constructed by restriction enzyme-mediated integration(REMI) technique in our laboratory. The suppression subtractive hybridization library was constructed using the mutant strain and wild strain with significantly different toxin production level. The genes differently expressed between those two strains were screened. The expression level of five genes chosen from differently expressed genes were tested and compared at different growth stages between the two strains by Real time quantitative PCR. Finally, adenosine kinase was selected to clone, obtaining cDNA full-length through RACE method and construct prokaryotic expression vector. The main results of this study are as follows:Firstly, according to Alternaria alternata suppression subtractive forward hybrid library previously constructed, about 1000 monoclonal white colonies by PCR validation were picked up with mutant as a driver and wild-type as a tester, and 819 short insertions were obtained. Then compared the spot differences on two membranes with two options: the one used positive sample hybrid probe, the other used reverse subtracted sample probe. Further significantly different spots were sequenced into ESTs which were classified. As a result,69 significantly different genes were screened between hybridization blots from a positive subtracted library. Most of the genes were homologous to Pyrenophora tritici-repentis. The genes varied and involved in energy and lipid metabolism, carbohydrate transporter pathway, coenzyme metabolism and ribosomal structure biosynthesis forming a wide range of metabolic networks related to the strain growth, toxin production, pathogenicity and so on. In addition, about 20% of unknown genes included.Furthermore, five genes related to toxin metabolism were chosen to do Real-time quantative PCR. The expression level differences of those genes in various growth stage were compared between mutant and wild strains to elucidate the relationship to toxin metabolim. The results showed that there were significant differences in the expression of the five genes in all growth stage. The expression level of thiazole biosynthesis enzyme in the wild strain reached the maximum at the sixth day, which was 11 times higher than the mutant but went down at seventh day. It may affects sporulation. The expression level of C-4 methyl strol oxidase exhibited a stepwise increase in wild type, but the mutant did not express basically. It can maintain the normal metabolism of Alternaria alternate during the entire growth process. The expression of adenosine kinase at the third day reached and maintained maxium level which was about 18 times higher than the mutant, until the seventh day decreased slightly. Compared to the other four genes, the overall expression level of this gene was the highest. The gene may influence toxin amount and virulence of mycelia by affecting the ATP metabolic balance. Phosphoglycerate kinase maintained 10 fold higher than the mutant since the third day. It involved in glycolysis metabolism, and affected the mycelium differentiation. The expression of sphingomyelin ceramide inositol hydroxylase gene in wild type changed irregularly and reached the highest level at the seventh day, which may affects sporulation.Finally, adenosine kinase gene with significantly higher expression level in the wild was thoroughly studied. The full length of 1342bp of ADK cDNA was sequenced by RACE method. The gene contains 915bp of complete open reading frame, encoding 304 amino acids. Its ends included 136bp of 5’-untranslated sequence and 285bp of 3’-untranslated sequence. The bioinformatic analysis showed that it has a high homology with other fungis, and 89% are homology with Pyrenophora tritici-repentis. ADK belongs to the ribosomal kinase protein family, which has typical combination of locial and conserved domains. Subsequently, ADK subcloned into the expression vector pET-28a (+), transferred to E.coli BL21(DE3) and lmM IPTG induced for expression. The protein with His-tagged about 38kDa was obtained. This protein presented in inclusion bodies through purification with 6 x His tag, and then exhibited good immunogenicity.