Selection And Validation Of Reference Genes And Conidiation-Related Genes Based On RNA-seq In Pandora Neoaphidis

Pandora neoaphidis,one of important Entomophthoralean fungi,is an obligated aphid pathogenic fungi that causes epizootics in aphid populations worldwide and has great potential for use in biological control applications.Because of its complex biological characteristics,P.neoaphidis and its aphid host were important models for studying on the “Insect-Fungi interaction” and host specificity between them.However,due to the molecular studies on the Entomophthoralean fungi,especially P.neoaphidis remain poorly understood,the potential genes of interest about fungal pathogens and biocontrol application are severely limited.Here,we carried out RNA-seq in hyphae and primary conidia by second-generation sequencing platform Illumina Hiseq2000,analyzed systematically the differentially expressed genes,to select the reference genes accoding stable expression analysis,to explore the ontology and pathway of gene expression,to mine and validate the conidiation-related genes of P.neoaphidis.The results were as followed.1.RNA-seq for hyphae and primary conidia of P.neoaphidisUsing RNA-seq,we totally obtained 90,639 contigs and 49,138 unigenes,besides,we got a total of 3555 differentially expressed genes,of which 2294 genes are up-regulated and 1261 genes are down-regulated according to RPKM.Based on GO functional enrichment analysis of genes differentially expressed results,49,138 unigenes were noted to 47 function category,covering three categories: cellular component,molecular function and biological process;metabolic process,cellular process,cell part,organelle-related genes expressed actively.KEGG pathway enrichment analysis indicated that glycosaminoglycan degradation and carotenoid biosynthesis are the dominant up-regulated metabolic pathways among 22 metabolic pathways during the hyphae stage to the conidia stage 2.Selection and validation of reference genes and conidiation-related genes in P.neoaphidisThe expression of 13 classical candidate reference genes(18S rRNA,28 S rRNA,ACT1,ACTN1,HPRT,EF1,GAPDH,TBCE,LDHA,LSM1,HISTH4,ALG9,DMA2)were evaluated by quantitative real-time polymerase chain reaction(q RT-PCR)at different developmental stages(conidia,conidia with germ tubes,short hyphae and elongated hyphae).By using four algorithms,including ge Norm,Norm Finder,Best Keeper and Delta Ct,we calculated the expression stability of the candidate reference genes.The analysis of comprehensive ranking revealed that ACT1 and 18 S rRNA was the most stable expressed gene set throughout the developmental stages.Using Cuffdiff software for GO function and analysis of differently expressed conidiation-related genes,we totally got 16 candidate genes.Evaluated by qRT-PCR,the results demonstrated that their up-regulation and down-regulation were all consistent to the RNA-seq data.This findings were of great significance in gene expression analysis of P.neoaphidis;meanwhile,it provide a foundation for further stability analysis of reference genes in P.neoaphidis and other Entomophthoralean fungi.3.Gene Cloning of white collar protein(WC-1)and its expression under different light condition in P.neoaphidis 3.1 Molecular cloning and function analysis of white collar protein gene wc-1Partial cDNA sequences encoding white collar protein(WC-1),was screened form the RNA-seq datebase.Prior to all cDNA fragments were completely spliced into a full-length cDNA through DNAMAN software,the 5' and 3' cDNA ends were synthesized by Rapid Amplification of cDNA Ends PCR(RACE PCR)using the target-specific primer pairs.The complete cDNA sequence of wc-1 contains a 2121 bp open reading frame and included a 5' untranslated region of 221 bp(UTR)and a 3' UTR of 389 bp,which encoding a protein of 706 amino acid residues with a predicted molecular weight of 78.62 k Da and theoretical isoelectric point of 6.49.The photoreceptor WC-1,which contains a special PAS domain,called LOV from “light,oxygen and voltage”,and had a DNA-binding zinc-finger domain.Phylogenetic analysis showed that the deduced amino acid sequence of wc-1 was closely matched to Fusarium pseudograminearum.3.2 Light-irradiation evaluation for conidiation of P.neoaphidis and expression of wc-1 conidiation-regulating gene.Irradiated by different wavelengths of light(i.e.,white,red,green,blue,yellow,black),the sporulation of hyphae of P.neoaphidis has significant difference.Results illustrated that the sporulation amount with irradiation of blue light was significant higher than other lights e.g.,green light.Sporulation amount with irradiation of dark was lowest.Additionally,blue light could significantly improve expression of wc-1 gene in 10h-age hyphae after 7 h irradiation.This result was consistent with RNA-seq analysis on wc-1 expression.Conclusively,those above results were obtained from the RNA-seq database and will provide good foundation for further studying the conidiation mechanism and other biological mechanisms of P.neoaphidis.Moreover,studies might lay the important scientific theory for exploring complex biological characteristics of P.neoaphidis,such as host specificity,also,might provide helpful promotion for comprehensive pests management and the field application of Entomophthoralean fungi,especially P.neoaphidis.