Screening And Functional Analysis Of The Genes Related To Chlamydospore Production In Clonostachys Rosea 67-1

Clonostachys rosea is an important biocontrol fungus that has shown great potential fighting against various plant fungal pathogens such as Botrytis cinerea,Sclerotinia sclerotiorum and Rhizoctonia solani.The fungal biocontrol agents composed of conidia are relatively unstable and have a short shelf-life.Chlamydospores are a specific resistant structure,which are of great significance in commercialization of biocontrol fungi.However,the research on the genes realated to chlamydospore production in biocontrol fungi is quite few.To invesigate the genes associated with chlamydospore formation in C.rosea,a highly efficient isolate 67-1 that originally obtained by our lab was selected,and the transcriptome of 67-1 during chlamydospore generation was sequenced and analysed.The reliable reference genes were identified to normalize gene expression during 67-1 sporulation.Gene knockout was conducted to verify the functions of the genes encoding hexokinase,polyketide hydroxylase and aldehyde dehydrogenase during chlamydospore formation.The study provided insight into the mechanisms underlying chlamydospore formation.In order to obtain genes related to chlamydospore from C.rosea 67-1,at 36 h and 72 h,we sequenced the transcriptome of 67-1 during chlamydospore generation,and obtained differentially expressed genes via high-throughput sequencing and quantitative real-time PCR.A total of 549,661,174 clean reads were obtained.At 36 h,67 and 117 unigenes were up-and downregulated in C.rosea during chlamydospore production,compared with the control for conidiation,and 53 and 24 genes were up-and downregulated at 72 h.GO classification suggested the differentially expressed genes were related to cellular component,biological process and molecular function categories.A total of 188 metabolism pathways were linked to chlamydospore production by KEGG analysis,among which metabolic pathways,biosynthesis of secondary metabolites,and metabolism to environments were dominant.16differentially expressed genes were selected and verified by quantitative real-time PCR,and the expression profiles of all 16 were consistent with the transcriptome data.Reference gene is essential for the quantification of gene expression.To screen the reference genes of C.rosea 67-1 during chlamydospore production,9 reference genes,actin?ACT?,elongation factor 1?EF1?,glyceraldehyde-3-phosphate dehydrogenase?GAPDH?,ubiquitin?UBQ?,ubiquitin-conjugating enzyme?UCE?,histone?HIS?,RNA polymerase II CTD phosphatase Fcp1?RPP?,TATA-binding protein?TBP?and succinate-semialdehyde dehydrogenase?SSD?were selected and cloned from 67-1,their expression stability during chlamydospore formation was determined using quantitative real-time PCR and assessed using the software geNorm,NormFinder and BestKeeper,ACT and SSD were selected to normalize gene expression during chlamydospore production in C.rosea 67-1.By tanscriptome data,we obtained hexokinase encoding gene HXK67,polyketide hydroxylase encoding gene PKH67 and aldehyde dehydrogenase encoding gene ALD67.Compared with the control for conidiation,at 36 h and 72 h,expression level of HXK67 upregulated 1.8-fold,89.1-fold,PKH67upregulated 11.8-fold,25.3-fold,ALD67 upregulated 16.1-fold,9.8-fold by quantitative real-time PCR.Bioinformatics analysis showed that the coding region length of HXK67 was 1602 bp,encoding a protein of 533 amino acids,and was closest to hexokinase-like from Acremonium chrysogenum with a homology of 63%.The coding region length of PKH67 was 1716 bp,encoding a protein of 571 amino acids,and was closest to polyketide hydroxylase from Colletotrichum gloeosporioides with a homology of 61%.The coding region length of of ALD67 was 975 bp,encoding a protein of 324 amino acids,and was closest to aldehyde dehydrogenase from Colletotrichum gloeosporioides with a homology of 49%.We constructed the knockout vectors of HXK67,PKH67 and ALD67 by homologous recombination and obtaind the deletion mutant?HXK67 by PEG4000-CaCl₂ mediated protoplast transformation.Phenotypes analysis showed that the growth rate of?HXK67 significantly higher than wild type,but no significant difference of chlamydospore formation and antagonism against Fusarium oxysporum.