| Nomuraea rileyi is environment-friendly biological control fungi that can infect a variety of Noctuidae(Leptoptera)pests.The conidia are the active ingredients of fungal insecticides,but the conidia spores on the conventional fungi culture medium is low,and the need for light and other factors limit the scale of the production and commercial applications.In our laboratory,we successfully induced the microsclerotium(MS)of Nomuraea rileyi by liquid fermentation.MS is a kind of dormant body structure formed by the large number of hyphae gathered in the specific condition.Due to its low cost,shelf-stable,resistance to adverse environments,good prevention effect and growth process without light,so it provids a new source for Nomuraea rileyi insecticides.Our previous laboratory works has confirmed that the formation of MS is a process of oxidative stress.At the same time,the of microsclerotium formation is accompanied by the precipitation of pigments,the production of reactive oxygen species(ROS)and the up-regulation of a large number of oxidative stress-related genes.The study of this regulatory pathway can provide an important theoretical basis for the development of highly virulent Nomuraea rileyi biocontrol agents.In this study,two up-regulated Cu/Zn-sod genes were screened from the established N.rileyi transcriptome library,named Nrsod1 and Nrsod2.The Nrsod1 and Nrsod2 gene function explored by gene knockout method.The main results are as follows:(1)Cloning and sequencing of sod family genes of N.rileyiWe successfully get the gene of Nrsod1 and Nrsod2 by PCR amplying.Sequence analysis showed that the open reading frame(ORF)of Nrsod2 gene was 915 bp,which encode 304 amino acids.The open reading frame of Nrsod1 gene was 564 bp,that encodes 187 amino acids and contain one intron with a length of 75 bp.The phylogenetic analysis was carried out by homology comparison and phylogenetic tree.The results showed that sod amino acid sequence encoded by the two genes was homologous to the sod of Metarhizium anisopliae and Metarhizium anisopliae,the homology were above 92%.The amino acid sequence of the sod supernumerine has conservative functional domain.(2)Construction of the knockout vector and Screening of transformants mutants of sodThe full-length sequence of the Nrsod1 and Nrsod2 genes and the 5 'and 3' flanking sequences were amplified by FPNI-PCR.The flanking sequences were recombined into the Pzp-Ptrpc-Hph-Knock vector to construct the knockout vector Pzp-hph-Nrsod1 and Pzp-hph-Nrsod2.The constructed knockout vector was transferred into Agrobacterium tumefaciens and was co-cultured with blastospore of the N.rileyi,the transformants were identified by PCR and analyzed by sequencing.We get the transformants were named ? Nrsod1 and ? Nrsod2.(3)Phenotypic analysis of mutants of ? Nrsod1 and ? Nrsod2The phenotypic analysis of mutants showed that: Compared with wild type,the time from yeast to mycelium of ? Nrsod1 and ? Nrsod2 delayed by 1.5 days.The yield of spores found that,?Nrsod1 and ?Nrsod2 reduced by 21% and 54%,respectively.The effection of mutants on germination rate and growth rate were not significant difference.Stress resistance analysis showed that the mutants showed different growth defects,and the ?Nrsod2 was more obvious,particularly methylenaphthoquinone completely inhibits its growth under the condition of salt stress,Congo red stress and oxygen stress,;The two mutants did not affect the tolerance of conidia to high and low temperature stress.The conidial germination rate of the mutant under UV irradiation was significantly lower than that of the wild type,The impaction of microbialsclerosis analysis found that: The microbial biomass of the two mutants decreased by 34.6% and 51.5%.The number of microflora decreased by 45% and 72.5%.Microscopic observation found that ?Nrsod1 and ?Nrsod2 microsclerotium formation delay,sclerotia reduced,specially,? Nrsod2 sclerotia irregular shape.(4)Analysis of gene expression patterns of ? Nrsod1 and ? Nrsod2Analysis of gene expression patterns showed that:Nrsod1 and Nrsod2 were up-regulated at 3d(microsclerotium formation stage),indicating that Nrsod1 and Nrsod2 were involved in the development of microsclerotium.And Nrsod2 in 5d(micro-fungi mature)again raised expression,indicating that Nrsod2 involved in whole process of microsclerotium.Nrsod1 and Nrsod2 gene interrelationship study found that: When the Nrsod1 gene was absent,the expression of Nrsod2 gene was significantly decreased,and the Nrsod1 gene expression was significantly decreased when the Nrsod2 gene was absent.The deletion of Nrsod1 and Nrsod2 genes does not stimulate the activation of the other gene,the absence of a gene inhibits the expression of another gene.sod gene and upstream and downstream genes related to the relationship research found Nrsod1 and Nrsod2 not only play an important role in the formation of micro-sclerotin,but also affect the formation of MS by regulating the activity of NOX complex and CAT family-related genes.(5)Toxicity determination of the sod mutantsThe results of the toxicity determination of the sod mutant were shown: mutant LT50 was significantly delayed compared with wild type by surface infection.The LT50 of Nrsod1 and Nrsod2 genes was 8.5d and 10 d,respectively,and the wild type LT50 was 7d,the LT50 of the Nrsod2 mutant was significantly delayed compared with the wild type;After 14 d,wild-type infection of Spodoptera litura all died,Nrsod2 mutant survival rate was 30%.The above results indicate that the spore virulence of Nrsod2 deletion mutant is significantly reduced.In summary,we constructed the mutant strains of Nrsod1 and Nrsod2 genes by Agrobacterium tumefaciens-mediated transformation.It proved that the Nrsod1 and Nrsod2 genes played a different role in regulating sporulation,growth and development,coping with stress,two types of transformation,and microbial regeneration.The qPCR study confirms that two genes affect the formation of MS by regulating the activity of NOX complexes and CAT family-related genes;Nrsod2 affects virulence. |
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