| Metarhizium plays an important role in the pests control,and it has been widely used in locust control.Metarhizium robertsii as a model creature of entomopathogenic fungi has played an important role in studying genetic regulatory mechanism,pathogenesis and biological control of entomogenous fungi.For many years,the researcher have committed to study the molecular mechanism about pathogenicity and resistance of M.robertsii,and have made some significant achievements;but as biocontrol fungi,the M.robertsii have the following disadvantages,including affected by the environment,slow speed of kill of insectsand so on,which restricts the further large scale of its application.Transcriptome sequencing is through the two generation sequencing platform for rapid comprehensive access to a particular cell or tissue in a table under almost all transcripts and gene sequences,today,transcriptome sequencing technologies have been widely applied in the eucaryon including Metarhizium,but,there are few studies on the transcriptome of M.robertsii;using the RNA-Seq to resolve the certain biological problems,including alternative splicing analysis,optimizing gene structur,and the prediction of novel transcripts and genes.Therefore,this study takes M.robertsii as the research object.Firstly,in the four different conditions which about growth and development,multi-stress tolerance,infection and colonization and degradation to cultivate M.robertsii for total RNA and transcriptome sequencing;Secondly,to complete the annotation of the novel transcripts which from transcriptome sequencing datas by bioinformatics analysis;Furthermore,we select the gene small ubiquitin-related modifier from the novel transcripts having been annotated.By using the principle of homologous recombination,small ubiquitin related modifier gene for knockout,and then through the comparison between the phenotype and growth in knockout mutants and the wild-type strain resistance,sporulation and virulence of small ubiquitin related modifier to study the gene function of M.robertsii.The results are as follows:(1)The transcriptome sequencinganalysis reveal novel transcriptsWe collected a total of 80,954,892 reads,after quality filtering,there were 46,674,896clean reads and the remaining 28,251,957 reads were mapped to the M.robertsii genome using TopHat.In this study,we compared the assembled transcripts of the sequence and the transcripts already present in the reference genome,and further predicted the novel transcripts.Findings:a total of 769 new transcripts were found,77 of which were predicted to code proteins.BLAST analysis and CDS(Coding sequence)searching of the 77 new transcripts which were predicted to code proteins to complete their functional annotation,at last,selecting the new transcript NovelTr-351 which be coding SUMO protein.There have been a lot of reports:SUMO is a critically important posttranslational protein modification which similar to ubiquitin,it not only can to negative regulation of transcription factors,but also be done on the activation of transcription.These could be response to environmental stimuli,however,thus it can be seen SUMO occupies a unique position as a kind of post-translational protein modification.It play an important roles in the control of almost all of the life processes,which including transcriptional regulation,nuclear transfer and maintaining genome integrity,besides useless protein degradation pathway,in order to the body’s normal operation.And some studies have shown that the ubiquitin-proteasome pathway can enhance the capacity of plant growth and adversity.Therefore,this study further selects SUMO for further analysis of its possible biological functions.(2)The study of the small ubiquitin-related modifierWe obtained gene deletion mutant△SUMO by knockout,and get cp△SUMO.Further phenotypic analysis showed that:using the PDA culture medium,knockout strain,wild-type strain and complemented strain comparison,the results found that the knockout strain of mycelial growth rate and the state and the realization of chemical resistance and toxicity,there was no difference;but the knockout mutant of color than the wild type and revertant strain in terms of spore color;in addition,knockout mutant sporulation is thinner,and the sporulation was significantly lower than that of wild type and revertant strain,down 50%.This means SUMO regulates the spore production process in M.robertsii;but also the knockout mutant strain has significant difference in the tolerance of high temperature and ultraviolet,in the high temperature 45℃in 1h processing cases,the knockout mutant strain and gene complement strain have 35-45%drop in the spore germination rate;Under the deal with Uv 100μJ/cm~2,the knockout mutant strain and gene complement strain have 40-50%drop in the spore germination rate.So SUMO maybe have a significant regulation effect to adversity tolerance in M.robertsii.The finding is to research the new transcripts and SUMO biological function in M.robertsiiby different conditions,the research results will give us a compacted base to improve the tolerances to stressful ability of high temperature,ultraviolet and spores. |
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