The Expression Pattern Of Genes Related To Resistance To Phosphine Of Sitophilus Oryzae

Sitophilus oryzae is an important storage pest,threatening the world's grain production.With the constsant use use of fumigant aluminum phosphide,S.oryzae has acquired a certain PH3 resistance.In this paper,through transcriptome technology,severalresistant genes related to S.oryzae such as glutathione S-transferase,cytochrome P450,EST were analyzed and the transcriptome genetic information was obtained.At present,with the development of biotechnology,especially the extensive application of genomics,revealing the resistance of insects in nature at transcriptome level is gaining a huge interest among the researchers.Sequences were read at 22,128,957,and 6,562,957,738 nucleotides using the Illumina sequencing technique,and the PH₃ fumigation strain was sequenced and observed reads were 27,306,829 and 8,093,178,954 nucleotides.The transcriptome data were assembled into 42173 unigenes by assembling.The acquired Unigene was compared to Nr for BLASTX,with 16,610 similarities.Among the 17,335 Unigene,44.45%of Unigene had the highest homology with related genes of Dendroctonus ponderosae.Out of that 26.96%of Unigene were homologous to the genes associated with Tribolium castaneum.A total of 323 Unigene related to detoxification and metabolic resistance were identified by screening,including 28 glutathione S-transferase genes,58 of esterase and 68 of cytochrome P450.A total of 13 full-length sequences of the GSTs gene were cloned.Six genes were classified into the Epsilon family according to the similarity and evolutionary relationship of the sequence.Four genes were classified into the Sigma family and two genes were classified in the Theta family;whereas,the the reamining one was classified in the Delta family.11 genes were cloned into the CYP4 family according to the similarity and evolution of the sequence.Six genes were classified into the CYP3 family and the CYP mito family was further classified into two groups.Six EST gene of S.oryzae were cloned which belonged to three different families.In the differential expressed genes of S.oryzae sensitive strain and PH₃resistance strain,the genes of S.oryzaePH₃ resistance strain were up?315?and down regulated?535?.The resulting differential expressed Unigene was submitted to the NCBI protein database,and a total of 687 returned effective results were observed.The Unigene in these differential expressed genes was divided into 61 functional groups of"biological process","cellular component"and"molecular function"in the GO classification.The COG classification annotated 221 differential expressed genes Unigene.Meanwhile,the results of differential expressed genes were classified according to the types of KEGG.The S.Oryzae sensitive strains of different concentrations of phosphine fumigation and fumigation treatment at different time were investiagted.The concentration of two genes was significantly higher than the other two concentrations by the concentration of LC₃₀,and the relative expression of the four GSTs genes was significantly higher than that of the control group and the LC₃₀,LC1₀ treatment group,and the relative expression of the three genes was significantly lower than that of the control group.The relative expression level of 7 GSTs genes increased gradually with the increase of fumigation time.The relative expression of the SoGSTd1 gene in the fourinstar stage was the highest in the relative expression pattern of the GSTs gene in the different developmental stages,and the relative expression of the SoGSTe1 gene in the adult stage was the highest.The relative expression of the SoGSTe2 and SoGSTe5 genes in the 4th instar stage was the largest,and the relative expression of the SoGSTe6 gene in the three instar larvae was maximum.13 GSTs genes of S.oryzae were differently expression in different tissues.The relative expression level of13 GSTs genes in midgut,fat body or malpighian tubules in S.Oryzae was significantly higher than that in the head and cuticle.The relative expression level of11 genes in the midgut,fat body or malpighian tubules was higher than that in the head and cuticle.In the EST gene,the relative expression of SoEST1 gene increased with the increase of the concentration of phosphine fumigation,and the relative expression of SoEST2 and SoEST5 gene by LC₃₀0 fumigation was significantly higher than that of the control group.Six EST genes were analyzed at different developmental stages.The relative expression level of SoEST1 in adult and pupal stage was significantly higher,and the relative expression level of SoEST3 gene in adult stage was the highest.In different tissues,the relative expression of SoEST1 gene in martensitic tube was the highest;the relative expression of SoEST3 gene in the midgut was the highest;the relative expression of the SoEST5 gene in the fat body was the highest,and the relative expression of the SoEST6 gene in the midgut was the highest.With the P450 gene in the S.Oryzae,LC₃₀0 concentration of phosphine fumigation by 24h,the relative amount of gene expression was significantly higher than that of control.The relative expression of seven P450 genes of S.oryzae was increased with the increase of phosphine fumigation exposure time.At different developmental stages,the relative expression level of five P450 genes in adult and four instar larvae was significantly higher;while,four genes were the highest in adult stage.In different tissues,four P450 genes displayed the highest expression in the midgut,and the three gene showed the highest expression in malpighian tubules.