Functional Analysis Of Triglyceride Synthase DGAT2 In Helicoverpa Armigera

Triglyceride is one of the indispensable nutrients in insect body,which affects the whole growth and development stage of insect.Diacylglycerolacyltransferase(DGAT),a substance that can directly participate in the synthesis of triglycerides,plays an important role in the physiological mechanism,but there is little research on insect DGAT2 gene.In this study,Helicoverpa armigera,the important agricultural pest,was selected as the research object.And we screened out the DGAT2 gene of H.armigera by bioinformatics analysis,and quantitative Real-time PCR analysis.Then,its function was verified by CRISPR/Cas9 gene editing and biochemical analysis.The main results are showed as follows:We obtained the related conserved domain from the H.sapiens DGAT2 gene in NCBI.We compared conserved domains and established the phylogenetic trees among28 species,such as Helicoverpa armigera,Drosophila melanogaster,Tribolium castaneum,Mus musculus and Homo sapiens,and we screened the Harm XP＿021192765.1 gene of H.armigera.We analyzed the spatial and temporal expression pattern of this gene,and the results showed that this gene is highly expressed in the third and fourth instars of larvae.The expression level of the third instar is 34.86-fold that of the egg stage,23.61-fold that of the first instar,3.27-fold that of the second instar,1.1-fold that of the fourth instar,3.87-fold that of the fifth instar,and 110.4-fold that of the pupal stage.This gene was highly expressed in the intestinal tract of larvae,but hardly expressed in other tissues.These results lay a foundation for the function verification of genes.We used the CRISPR/Cas9 gene editing technology to knock out large fragments of DGAT2 gene.We found that the dysfunction of this gene had some delayed effects on the fourth instar and pupal development of G0 generation.The developmental duration was delayed by 15.8% and 6.5% respectively,and the eggs produced by F2 generation adults could not hatch.According to the measurement of intestinal indexes of H.armigera,there was no significant difference in the contents of monoglyceride and diglyceride in the intestine of mutant,and the mass of intestinal tissue did not change.Triglyceride content decreased by 57%,and mass of intestinal tissue was not affected.The DGAT2 enzyme inhibitor experiment showed that the triglyceride content and tissue mass in the intestine of the mutant individuals were not affected by the inhibitor,and the triglyceride content in the intestine of the wild type was significantly decreased by 47.6%,and the dry weight of the intestinal tissue was not significantly different.The results showed that the target gene was involved in the synthesis of triglycerides,and did not have the function of MGAT.Moreover,the enzymatic activity could be significantly inhibited by NEM,a specific inhibitor of mammalian DGAT2.Therefore,the gene was identified as DGAT2 by the above comprehensive analyses.In conclusion,this study identified the triglyceride synthesis gene DGAT2 of H.armigera for the first time,and verified the related functions of this gene involved in the pathway of triglyceride synthesis,which further improved the nutrient synthesis mechanism of H.armigera,and provided a new target for pest control of H.armigera.