The Study Of Phosphine Resistance Mechanisms In The Cryptolestes Ferrugineus

Rusty grain beetle,Cryptolestes ferrugineus is an important pest worldwidly which develop resistance quickly against phosphine.However,the lack of genetic information on this organism is an obstacle to understand the mechanisms of resistance.So,the high-throughput sequencing technology was employed to sequence the C.ferrugineus transcriptome and Digital Gene Expression Tag Profiling in order to expand the gene information resources of the species and lay a molecular foundation for future study of phosphine resistance of C.ferrugineus.The main results are as follows:1.C.ferrugineus used in this study were collected from Zhangjiagang,Suzhou;Chengdu,Sichuan;Taicang,Suzhou.The value of LC50 was 0.079 mg/L、0.126 mg/L、19.904 mg/L,respectively.Accroding to the LC50 value of susceptible strain given by the United Nations,Rf value of each strain can be calculated: Rf value of ZJGCF is 7;Rf value of CDCF is 12;Rf value of TCCF is 1,809.2.Sequencing by Illumina Hiseq 2000 platform,total output 4.6GB data.After assembling,a total of 47,006 unigenes were obtained with the total length of 51,214,924 bp,among which 28,491,14,036,23,667,21,320,12,576,14,930 unigenes were annotated via BLAST querying against current databases including Nr,Nt,Swiss-Prot,KEGG,COG,GO(E-value <0.00001).Prediction coding sequence(CDS)were 34,631 in all,the number of CDS which could be annotated by protein database was 28658 and CDS which could be predicted were 5,973 eventually.A total of 115,410 unigenes were annotated by GO database.Among the three ontologies,biological process took up the largest group.The main process of it was cellular process and single-organism process.Followed by cellular component was mainly involved in cell,cell part and organelle.Molecular function had the least number of annotations,the top two largest sub-function annotation group were binding and catalytic activity.There were 28,976 unigenes annotated to COG database,which were classified into 25 categories.They were main related to “general function prediction only”,“Replication,recombination and repair”,and Transcription.KEGG database showed that 29323 unigenes participated in 252 pathways,mainly enriched in metabolic pathways and regulation of actin cytoskeleton.4,836 SSRs and 120,212 SNPs were identified to provide an effective tool for the study of population genetics.3.By sequencing the Digital Gene Expression Tag Profiling of susceptible strain and phosphine-resistant strain of C.ferrugineus.We respectively obtained clean reads between 11.7 and 12.6 million.Among those genes,there are 37,792 differentially expressed ones,including 23,802 up-regulated genes and 13,990 down-regulated genes.Meanwhile,up to 54 genes were expressed significantly differently,including 28 up-regulated genes and 26 down-regulated genes(FD≤0.001 and log2Ratio≥1.Moreover,significant enrichment of 54 GO terms and 36 Pathways were identified by GO function significant enrichment analysis and Pathway significant enrichment analysis,respectively.Further clustering the differentally expressed genes with similar function by the expression pattern cluster analysis,many of genes related to phosphine-resistant are likely contained in these results.Comparing the gene expression between the phosphine resistant and susceptible strains of C.ferrugineus,we can find that cuticular protein took a lager proportion of up-regulated gene annotation,studies had shown that it is generally involved in the regulation of epidermal penetration of insecticides in the mechanism of toxicity.Cytochrome b,ATP synthase subnit and NADH dehydrogenase subunit were three kinds of protin that had clear annotations among down-regulated genes.Refered to the literature,they are all the critical genes involved in cell respiration in the mitochondria.After a long time with phosphine fumigation,the significant decrease in expression of these genes indicated that mitochondrial have inextricably relationships with the formation of phosphine resistance mechanism,studying the key regulatory proteins is benefitial to clarify the phosphine resistance mechnisms.4.Unigenes obtained from the transcriptome of C.ferrugineus and the resistance-related genes(detoxification enzyme)of Drosophial melanogaster in the NCBI were aligned and analyzed through phylogenetic analysis.They are P450 genes(68),GSTs genes(25),CarEs genes(10).Screening resistance associated genes and identifying their family classification by homology analysis will provide the basis for subsequent functional studies,but also will be conducive to clarify the mechanisms of phosphine resistance of C.ferrugineus.5.This is the first study to use the three algorithms(geNorm,NormFinder and BestKeeper)to validate a set of reference genes for normalizing gene expression by qRT-PCR in C.ferrugineus.Our results showed that γ-TUB + RPS13 were best normalizers to analyze gene expression profiles during the various developmental stages,and that SDHA + RPS13 + GAPDH could be ideal to analyze gene expression differences among different strains of C.ferrugineus.