Detection Of Phosphine Resistance Level Of Rhyzopertha Dominica And Excavation And Verification Of Resistance-related Genes

Rhyzopertha dominica is a kind of world-wide grain storage pest,which seriously threatens the safety of grain storage.Phosphine as an excellent fumigant is widely used all over the world.However,with the development and enhancement of resistance to phosphine,it has severely restricted its fumigation effect.Therefore,exploring the molecular mechanism of the resistance to phosphine is of great significance for effective prevention and treatment,the proposal of comprehensive management strategies,and the development of new medicaments.In this article,we started by clarifying the phosphine resistance levels of different strains of Rhyzopertha dominica,using high-throughput sequencing technology to sequence the transcriptome of Rhyzopertha dominica,obtain its genetic information,screen its resistance-related genes,and provide some theoretical data for the molecular mechanism of Rhyzopertha dominica phosphine resistance.The main findings were as follows:1.According to the method recommended by FAO for the determination of phosphine resistance,the phosphine resistance level of the Rhyzopertha dominica populations collected from 10 different areas was determined.The results showed that the LC₅₀values of the GX and LL populations were 57?g/L and 78.1?g/L,and the resistance coefficients were 7.1 and 9.7 times,belonging to low-resistant populations.The LC₅₀values of HG,YY,ML,WL and DC populations were 90.3?g/L,138.7?g/L,153.1?g/L,233.8?g/L,248.6?g/L,and the resistance coefficient were 11.3,17.3,19.1,29.2,31.7 times,belonging to the medium resistance population.The LC₅₀value of the LH population was 417.3?g/L,the resistance coefficient was 52.1 times,belonging to the high resistance population.The LC₅₀values of HH and XD population were 5,899?g/L and 9,389?g/L,resistance coefficients were 737.4 and1,173.6 times,belonging to extremely high resistance populations.2.By sequencing the transcriptomes of different strains of Rhyzopertha dominica phosphine,a total of 84,723,044 original data were obtained.After the original data were filtered,the total number of clean reads was 84,577,020.After assembly,a total of 47,379 genes were obtained.The length of N50 was 1,705bp,the maximum length was 35,796bp,and the average length was 952bp.Annotate all genes into the four major databases of NR,KEGG,COG,and Swiss Prot,and the numbers of genes were 25,623,22,753,16,912,and 19,262 respectively.Based on the analysis of inter group differences,significant difference genes were screened among the groups,including 369 significantly up-regulated genes and 364 significantly down regulated genes in the comparison between the control group of resistant population and the control group of sensitive population;123 significantly up-regulated genes and 473significantly down regulated genes in the comparison between the experimental group of resistant population and the control group of resistant population;and 123significantly up regulated genes and 473 significantly down regulated genes in the comparison between the treatment group of resistant population and the control group of resistant population.There were 800 up-regulated genes and 358 down regulated genes in protective coma group.Through go function analysis,we found that the most genes involved in biological process,mainly involved in cell process and single tissue process,followed by cell components,and the least genes involved in molecular function annotation.Pathway significance analysis showed that the differential genes were mainly enriched in metabolic pathways.The mechanism of phosphine resistance could be effectively explained by analyzing the function of genes with significant differences among groups3.Based on the results of transcriptome data,seven candidate internal reference genes were screened,and the expression levels of candidate internal reference genes were detected by real-time fluorescent quantitative PCR under the conditions of high temperature,low temperature,phosphine induction and different development stages.Meanwhile,their stability was analyzed and evaluated by ge Norm,Best Keeper and Norm Finder.The results showed that 18S r RNA+?-TUB was the most stable gene combination,actin5c and Pol II were the most unstable genes,and GADPH+18S r RNA genes combination could be used as the internal reference gene at different development stages,and?-TUB was the most unstable gene.The suitable internal reference gene could lay a foundation for further study on the mechanism of Phosphine Resistance of R.D.4.Real time PCR was used to verify the expression level of 9 candidate genes.The relative expression levels of different genes in GXC,GX4h,XDC,XD4h and XDT groups were detected,and then compared with the data of transfer group.The results showed that the expression trend of 8 genes was the same as that of transcriptome sequencing.The expression of EGT,GSTS1 and LIPK were all on the rise,and the expression of LIPN,Deltatry,CHYM1 and TRYP3 were down regulated,and the expression of LIP1 between xd4h and XDT group decreased,and that between XDC and GXC group was on the rise.But the gene UGT1A3 was not significantly down regulated between GXC and GX4h group,but there was no significant difference.