Discovery And Functional Analysis Of Cholesteryl Esterase Gene In Helicoverpa Armigera

Insects are sterol heterotrophic organisms and can only meet their own sterol nutritional needs by feeding.In addition to free sterols,plants also have a large amount of steryl esters.However,it is still unclear how insects utilize steryl esters.Helicoverpa armigera,an important agricultural pest in China,has imposed a seriously negative effect on agricultural production in our country.This thesis took the cotton bollworm as the experimental subject,and investigated whether the insects can utilize steryl esters.The main results are showed as follows:1.We screened out a steryl ester,cholesteryl oleate,which can be utilized by the cotton bollworm through biological analysis.2.We used high-throughput sequencing and bioinformatics analysis to screen out three candidate genes,namely Ha OG200551,Ha OG200552 and Ha OG200589.3.The expression patterns of the candidate genes were analyzed by q PCR,and the results showed that the relative expression of three candidate genes were all significantly higher in the midgut than in cuticula,and the cholesteryl oleate treatment could induce their expression.Among the three candidate genes,the spatio-temporal expression patterns between Ha OG200551 and Ha OG200589 in different instars were almost consistent,while Ha OG200552 showed a different pattern.4.CRISPR/Cas9 genomic editing technology was employed to explore the function of Ha OG200589.The results showed that the growth of the mutants was significantly slower than that of the control group,and the amount of steryl ester in its frass was significantly higher than that in the control group.5.The yeast signal peptide screening was used to verify the secretory function of the signal peptide in the three candidate genes.The results showed that all the signal peptides have secretory function,which indicated that the three genes all encode secretory proteins.6.The prokaryotic expression system was used to express Ha OG200589 in vitro and the purified product was obtained,which laid a solid foundation for further in vitro experiments.Overall,this study elucidated that the cotton bollworm could utilize steryl esters.We found that three cholesteryl esterase genes were likely involved in the metabolism of steryl esters in the diet.