Molecular Mechanism Of Cholestanone Metabolism Mediated By Aldo/keto Reductase AKR2E9 In Mythimna Separata

Cholesterol,the dominant sterol in most animals,participates in several key physiological processes,such as the composition of cell membrane and the precursor of ecdysone.Insects are sterol-auxotrophic organisms so they typically obtain sterols from food.However,cholesterol is rarely found in plants above trace levels,and insect herbivores have to convert phytosterols into cholesterol.Insect herbivores are co-evolved with plants in the metabolism of secondary metabolites,and the capacity of metabolizing phytosterols.Insects vary largely in the efficiency of dietary sterols absorption and metabolism,as well as their demand for sterols.These characteristics and differences in sterols metabolism are important in pest control.Some atypical phytosterols,such as stanols and ketone-steroids,have similar structure as cholesterol,but cannot be metabolized to cholesterol.Additionally,these sterols may interfere with the normal function of cholesterol so that they can cause deleterious effects on development and reproduction,even leading to death,and has great potential in pest control.Currently we know very little about the sterol nutritional characteristics of M.separata,as well as the genes involved in sterol metabolism.In this study,we underwent experiments based on a semi-synthetic artificial diet to investigate the sterol nutritional characteristics,including sterol demand,adaptability to different sterols and the metabolism capacity of atypical sterols.We screened the gene involved in the metabolism of cholestanone and further demonstrated the importantce of this gene in the metabolism of cholestanone in vivo and in vitro.The major findings of this study are described as follows:1.The adaptability of M.separata to different sterols/steroids.In this study,we found that the amount and species of dietary sterols would affect M.separata on development and survival.The diet containing 1 mg/g cholesterol（1C）can successfully allow the larvae to pupate but the performance decreased when the concentration was reduced.The adaptabilities of M.separata were not same on different sterols including cholesterol,sitosterol,stigmasterol,cholsteranol and cholsteranone.Moreover,the diet containing 1 mg/g cholestanol（1A）successfully supported all the larvae to pupate while the cholestanone at the same concentration（1K）caused all larvae to die in the early stages,e.g.,the 1^st or 2^nd instar.We then provided larvae with a mixed diet containing cholesterol and cholestanone.The results indicated that the addition of cholestanone could significantly improve larval performance when the cholesterol was deficient in the diet（<1C）.But when cholesterol was sufficient in the diet（1C）,the addition of cholestanone（0-2K）would not affect larval survival.Two metabolites,cholestanol and epi-cholestanol,from cholestanone were recovered in their bodies,indicating that cholestanone can be metabolized into the lower toxic sterols.In summary,the larval development and survival can be affected by the content,type and ratio of dietary sterols,and M.separata was capable of detoxifying cholestanone.2.Identification and in silico analysis of the gene involved in cholestanone metabolism in M.separata.In this study,we firstly found eight genes which might relate to the metabolism of cholestanone of M.separata according to the previous study on Helicoverpa zea and de novo assembly transcriptome analysis of M.separata.The eight genes were classified to three clades,aldo/keto reductase（AKRs）,ubiquitin-conjugating enzyme,and enoyl-（Acyl carrier protein）reductase.An AKR,Mse29229,was highly expressed in M.separata gut and significantly up-regulated by cholestanone.The gene was named AKR2E9 according to the fact that the high-confidence homology with AKR2E subfamily by establishing dendrogram and multiple sequence alignment.AKR2E9 has a characteristic fold and a（α/β）₈-TIM barrel,the structure that typically occurs in AKR family members.In the center of the barrel,there is a catalytic tetrad（Asp70,Tyr75,Lys104,and His137）.We further found the expression level of AKR2E9 protein was significantly increased in cholestanone-fed larvae than cholestanol-fed larvae by the specific antibody.Moreover,AKR2E9 protein was detected in the intestine tissue,but not in either the intestinal lumen or the oral cavity,suggesting that the reduction of cholestanone by AKR2E9 occurred in the intestinal cells.3.Prokaryotic expression and in vitro functional validation of AKR2E9.A large amount of soluble His-AKR2E9 protein was expressed in vitro by prokaryotic expression system,and then concentrated to a purified protein with the concentration of 10mg/g.Western blot analysis showed that the M.separata AKR2E9 protein was successfully expressed by the specific antibody.Aldo/keto reductase（AKRs）is a reductase that catalyzes the NADPH-dependent reduction of carbonyl into hydroxy.We established the reaction system including cholestanone as the substrate,and NADPH as the cofactor.Cholestanol was detected as the sole metabolite of cholestanone by GC-MS technology.These results showed that the recombinant protein His-AKR2E9 expressed in Escherichia coli was able to convert ketone into the corresponding stanol in vitro.4.Optimization of the application of the CRISPR/Cas9 system in M.separata.In this study,we used the gene NPC1b which contained no intron as the target gene,to optimize CRISPR/Cas9 system for M.separata.We recorded the egg-laying characteristics,and found that females gave their highest egg production on days 5-7,and the eggs produced on days 6-8 had highest viability.Moreover,female moths laid most of their eggs in the first3 h of the dark period.Different solutions were tested to separate eggs from the sulfuric acid paper,and 0.1%Na Cl O was found to be a more appropriate solution with lower effect on egg viability.Importantly,we must apply some water on egg surface when injecting.Otherwise,the egg broke by the injection and failed to hatch.This is a special procedure for the application of the CRISPR/Cas9 system in M.separata,which,as far as we know,was different from other lepidopteran insects.Besides,a humid condition is also required for egg incubation.Freshly-laid eggs should be used for injection as soon as possible,or the hatching rate and the editing efficiency decreased.5.CRISPR/Cas9-mediated function analysis of the AKR2E9 in M.separata.By comparing the structure of the orthologs in other lepidopteran insects which have their genomes,we identified the exons/introns for M.separata AKR2E9.We adopted a long deletion strategy using dual sg RNAs and then successfully established homozygous mutations,AKR2E9^-/-.There was no phenotype difference between AKR2E9^-/-mutants and the wild-type insects when they were reared on the cholestanol diet.However,the performance of AKR2E9^-/-mutants was greatly reduced on the cholestanone diet.GC-MS analysis showed that the levels of the metabolites of cholestanone were reduced while cholestanone was increased in the body of AKR2E9^-/-larvae in comparison to that in the wild-type insects.Compared with the wild-type,cholesterol oxidase can significantly reduce the performance of AKR2E9^-/-larvae on the cholesterol diet but not on the cholesterol oleate diet,which further demonstrated the essential role of the AKR2E9 gene in the metabolism of ketosteroids in M.separata.In summary,we created an AKR2E9^-/-mutant line to investigate the role of AKR2E9 in the metabolism of cholestanone,and the results suggested that it played critical role in metabolizing cholestanone in M.separata.In conclusion,we explored the sterol nutritional characteristics of M.separate for the first time,and elucidated the role of AKR2E9 in metabolizing and detoxifying ketone steroid.This work extensively investigated the sterol nutritional biology of M.separata and provided critical information for the application of CRISPR/Cas9 system in M.separata,and the results can help us to develop effective controlling strategies for M.separata from the sterol nutritional perspective.