Analysis Of Digital Gene Expression Profiles Of Phosphatase-sensitive And Resistant Lines In The Genus

The red flour beetle, Tribolium castaneum (Herbst) is widely distributed around the world in stored grain pests. T. castaneum infest stored grain by direct feeding, manure pollution and accelerating the moldy of grains. At present, phosphine becomes the best fumigants of storage in the world. Due to the unscientific use of phosphine, T. castaneum commonly generate different degrees of resistance to phosphine on a global scale. With the development of the world trade, the damage range is increasingly wide, and the resistance of phosphine becomes more and more serious. Previous studies on phosphine are starting on the one hand, and lack of systematicness and integrity. Gene expression profile of mRNA can research the transcription and regulation of gene in the cell in the overall level. The method considered the interaction between the environment and the genome, and provided effective research methods for the mechanisms of phosphine toxicology. And the completed T. castaneum genome, also provided a solid foundation from the overall levels for phosphine. In order to further understand the mechanisms of action of phosphine and the mechanism of insect resistance, T. castaneum is chosen as the object of this study. We using the high-throughput Illumina RNA-seq technology to construction the digital gene expression profiles of no fumigation susceptible strains (NF_S), no fumigating resistant strains (NF_R), fumigating susceptible strains (PF_S), and fumigating resistant strains (PF_R). After screening the differentially expressed genes of NF_R vs NF_S, PF_R20 vs PF_S20. PF_R20 vs NF_R and PF_S20 vs NF_S, we mapped the differentially expressed genes to terms in KEGG and GO databases, and made pathway and GO enrichment analysis. Based on the digital gene prodiling results, we make a further analysis of the respiration, antiosidant and detoxifying enzyme change of the untreated susceptible and resistant T. castaneum and phosphine-treated susceptible and resistant. Finally, fluorescence real-time quantitative PCR was used to vertify the expression level in four samples of six genes.1. The sequence data. After filtered the four sample produced 6,653,758 (NF_R)、 7,838,702 (NF_S)、6,675,764 (PF_R20) 和 8,257,496 (PF_S20) seqence of 53 bp.2. The screening of differentially expressed genes. The differentially expressed genes of NF_R vs NFS. PF_R20 vs PF_S20. PF_R20 vs NF_R and PF_S20 vs NF_S are 425,1400.559 and 975.3. Functional analysis of differentially expressed genes. (1) Functional analysis of differentially expression genes of NF_R vs NF_S can provide the differences of the susceptible and resistant. The GO functional enrichment of NF_R vs NF_S are associated with the function of mitochondrial. protenin metabolism, and chitin metabolism. The result of KEGG enrichment indicated that, the mainly difference are in the antioxidant. (2) The functional analysis of differentially expression genes of PF_S20 vs NF_S can provide the information of the toxicological mechanism of phosphine. The GO functional enrichment of PF_S20 vs NF_S indicated that phosphine have significantly effect on the function of mitochondrial. The GO and KEGG functional enrichment indicated that phosphine have signidicantly effect on sugar metabolism and protein metabolism. (3) The functional analysis of differentially expression genes of PF_R20 vs NF_R can provide the information of the mechanism of resistance. The GO and KEGG functional enrichment indicated that the degradation protein and chitin metabolism are changed. (4) The functional analysis of differentially expression genes of PF_R20 vs PF_S20 can provide the difference of the treated-resistant and treated suspectible. The GO functional enrichment incicate that the differences of PF_S20 and NF_S are mainly on mitochondrial function, sugar metabolism, antioxidation, protein metabolism and detoxification.4. Make preliminary analysis of the differences of respiratory, antioxidant and detoxifying enzymes of phosphine-treated susceptible and resistance strains. (1) Phosphine may promote glycolysis of the phosphine-suspectible and the citric acid cycle of phosphine-resistant. (2) After treated by phosphine, the expression of SOD, POD, and HSP of T. castaneum are up-regulated, and the fold of phosphine-resistant are more than phosphine-suspectible. (3) The analysis of detoxifying enzyme indicated that phosphine may have significant effect on the activity of monooxygenase of phosphine-suspectible; the expressions of GSTs of phosphine-resistant and phosphine-suspectible are up-regulated; the expressions of CarE of phosphine-resistant are up-regulated, and the the expressions of CarE of phosphine-suspectible are up-regulated and down-regulated.5. qRT-PCR verification. In order to confirm the reliability of digital gene expression profiling, we use fluorescence real-time quantitatibe PCR to vertified 6 genes screening from the result. The result of digital gene expression profiling is consistent with the fluorescence real-time quantitative results.All in all, the toxicological mechanism of phosphine is associated with the function of mitochondrial, the sugar metabolism and the protein metabolism. The mechanism of resistance is associated with that the phosphine-resistant can maintain the normal function of mitochondrial, sustain morma energy metabolism; degradation the damaged protein in time; the antioxidant ability is stronger than the phosphine susceptible.