Karyotype Analysis And FISH Analysis Of Infection Related Genes In Metarhizium Acridum

Fungi is a eukaryote, with the nucleus of the cell wall and the real, non-chlorophyll, parasiticor saprophytic way to survive; Typical fungus have both sexual reproduction and asexualreproduction, and produce a variety of forms with spores capabilities. Fungus and humanhealth has a great relationship: penicillin produced by Penicillium bacterial infections canbe treated, as well, the fungus will pose a threat to human health, data analysis, on humanpathogenic fungi, about300species, such as infant Candida Candida colitis, caused byTrichophyton tinea fungal infections are due to other causes; recent years, withbroad-spectrum antibiotics, immunosuppressants, anti-cancer drugs widely, carry out organtransplants, surgical techniques and catheter interventional treatment of other conditionscaused by pathogenic fungi systemic fungal disease is also increasing,emerging newpathogens, the disease has become increasingly serious, so the development of newantifungal drugs has become a priority.Metarhizium acridum is an insect host filamentous fungi, because of their geneticbackground has been studied well, and for people, animals and the environment areharmless, it can serve as an ideal biological fungal infection and pathogenesisstudies.Research and development of antifungal agents, it is necessary to study themechanism of fungal infection; Although genes have been associated with fungal infectionwere studied part, such as Pr1and Pr2protease genes, their ability to have a fungalinfection a regulatory role, but the role of these genes is not alone, but by the synergisticeffect of the process to achieve complete infection and produces biological effects. Clearthe infection-related genes on the chromosome location helps to understand, study thesepatterns of gene expression and its correlation function to lay the foundation for the studyof fungal infection from the whole gene level mechanisms, but also for the futuredevelopment blocking fungal infection antifungal provide a theoretical basis.Karyotype analysis is the basis of genetic research and functional genomics. Only inkaryotype analysis done on the basis of DNA can be labeled and purpose of gene mapping;currently karyotype analysis of fungi is difficult, due to the fungal chromosome smaller,shorter length, centromere, with the body, such as signatures are difficult to observethrough the microscope, existing methods for fungal karyotype studies are mainlytraditional optical microscope observation method and pulsed field gelelectrophoresis(PFGE), As the fungus chromosome small, so are the choice in aconventional optical microscope by observing fungal cells in metaphase, so as to distinguish each chromosome under the microscope, however also often underestimate thenumber of fungal chromosome; pulsed field gel electrophoresis because of its fast andefficient features and is widely used to detect different species karyotype analysis, but italso has some limitations, such as can not be directly observed chromosome morphology,gel electrophoresis separation only10kb to10Mb size of chromosomes,and when thenumber of chromosomes is very close to the size it can not be underestimated and thuspoints to the number of chromosomes by pulsed field gel electrophoresis to leave. Usingoptical microscopy karyotype analysis of fungi Neurospora crassa, Tuberaestivum,Erysiphe graminis, Erysiphe, Penicillium chrysogeum etc.; using pulse gelelectrophoresis karyotype analysis of fungi Nectria haematococca, Cochliobolusheterostrophus, Botrytos, Rhizoctonia solani etc.; therefore research and positioningM.acridum karyotypes associated genes will provide new research methods and theoriessupplement for research.In this study, M.acridum strains CQMa102for the study,the expression vector byconstructing Pgpd-H1-GFP and expression in M.acridum, thus the mitotic behavior ofdynamic could be observed in vivo; using a modified germ tube burst method forchromosomes are observed by conventional optical microscopy and chromosomekaryotype analysis, and by fluorescence in situ hybridization(FISH) of ribosomalRNA(rDNA) in chromosomes analyzed, concluded as follows:1M.acridum dynamic observation of mitosis in vivo1.1Constructing Pgpd-H1-GFP expression vector and successfully transferredM.acridumIn PK2-PB-EGFP vector based herbicide resistance with Bar(glufosinate) genes wereinserted into the composition of our promoter fragment and expression Pgpd fragment ofhistone H1, identified by recombinant PCR, restriction enzyme digestion and verified bysequencing successfully constructed Pgpd-H1-GFP vector; then make the method ofAgrobacterium-mediated transformation Pgpd-H1-GFP successfully transferred M.acridumand stable expression in vivo.1.2Screening M.acridum H1-GFP expression strain, and dynamic observation ofmitosis in vivoFluorescence microscopy confirmed H1-GFP successful presentation in M.anisopliae.H1-GFP localized in the nucleus of the chromosome, so that the chromosomescan be identified to bring the fluorescent label. With this transformation can observemitosis in vivo. 2M.acridum Ma102karyotype analysis2.1Improved M.acridum Ma102chromosome specimen methodBy optimizing the acid treatment time and increasing hypotonic and other steps,drawn M.acridum karyotype specimen preparation methods are as follows: Select freshM.acridum spores using1/4SDA liquid medium incubate8-10h, until after sporegermination grow germ be collected by centrifugation, and the use of methanol and glacialacetic acid(V/V=17:3)and fixed overnight, and were anhydrous ethanol and70%ethanolfixed, whose time was2h; hydrochloric acid dissociation method M.acridum cell wallprocessing, processing conditions for1M HCl60℃water bath8-14min; using ice-cold1×PBS hypotonic20min; staining dye used is the grilled slices Giemsa or DAPI, stainingtime was30min and10min.2.2Observation chromosomes of M.acridum Ma102by microscope and karyotypeanalysisObservation of the chromosomes of60specimens karyotype clear statistical analysis,the average number of Ma102M.acridum chromosome is10, Ma102average relativelength of chromosome chromosomes are16.3%,13.4%,12.7%,11.3%,9.9%,8.9%,8.2%,7.2%,6.3%,5.7%. Depending on the relative length of each chromosome karyotype drawa schematic diagram, observed Ma102relatively long prophase chromosomes,chromosome photos can be observed partial chromosome centromere and satellite andother structures.3Fluorescence in situ hybridization analysis(FISH) for rDNA of M.acridum.Fluorescence in situ hybridization analysis of rDNA in M.acridum.According to Roche of the kit instructions and Southwest University laboratory in situhybridization proces, using rDNA probes prepared by nick translation with M.acridumchromosome samples were hybridized, through a series of wash off and signal detectionprocessing, and ultimately by fluorescence microscopy to observe the hybridization signal,and photographed records and data analysis, Lay the theoretical foundation for the futurepositioning infection-related genes.