Identification And Functional Analysis Ofsterol Transporters And Metabolic Genes In Helicoverpa Armigera

Cholesterol is an essential nutrient for insects and it performs two important biological functions in insects.Firstly,it is the main component in cell membrane and plays an important role in maintaining the stability of cell membrane.Secondly,cholesterol acts as the precursor to sterol hormones(e.g.,ecdysone).Unlike mammals,insect cannot synthesize cholesterol de novo and must acquire cholesterol from food.In plants,however,phytosterols are the main sterols and cholesterol is rarely found.So many phytophagous insects need to convert phytosterols into cholesterol by dealkylation.Therefore,the genes involved in metabolizing and transporting sterols are important for insects.The acquisition or transformation of sterol will be blocked if the function of genes related to sterol absorption or metabolism were interrupted.This method which has important application value can be used as a pest control strategy.However,researches on the genes involved in sterol absorption and transportation is scarce in insects and the function of related genes are unclear.Two main aspects of research were conducted here.First,using the cotton bollworm as the model organism,the function of related genes was analyzed by qRT-PCR,fluorescence in situ hybridization,CRISPR/Cas9 and GC-MS.Second,genes involved in phytosterol metabolism were identified through high-throughput sequencing technology and the function of candidate genes were investigated by CRISPR/Cas9 and GC-MS.The main research results are showed as follows:1)In insects the phylogenetic relationship of these genes was unclear.In this study,we identified NPC1 genes in 39 insect species across 10 orders based on high-throughput sequencing data.Thirty of 39 species contain two homologs,NPC1 a and NPC1 b.The divergence of NPC1 likely occurred in the ancestor of insects.At the same time,we found that the loss of NPC1 occurred in 9 insect species,main in sternorrhynchan insects.Interestingly,unlike other NPC1 b and all NPC1 a genes,NPC1 b in the lepidopteran species investigated in this paper contained no introns.All NPC1 proteins are relatively conserved indicating that they may have the similar function.2)It is known that NPC1 b is involved in the intestinal absorption of sterols in fruit flies while NPC1 a is responsible for the intracellular transport of sterols.However,study on these two proteins in Helicoverpa armigera was never reported and their functions are unknown.We investigated the expression pattern of NPC1 genes and mainly explored the role of NPC1 b in sterol absorption.The temporal and spatial expression patterns of H.armigera between NPC1 a and NPC1 b were quite different.NPC1 a is widely expressed in tissues and stages.However,NPC1 b was highly expressed in midgut with no expression in hindgut and low expressed in other tissues.Results showed that NPC1 b was highly expressed in larval feeding stage and very low in egg and pupa.Low concentrations of cholesterol in food can promote NPC1 b expression.The modelling and molecular-ligand docking analysis indicated that NPC1b_N of H.armigera contained 10 amino acid sites(Gln73,Asn106,Lys76,Phe98,Ile102,Met105,Met164,Leu188,Ala189,and Pro190)interacting with cholesterol molecule.Six residues were identical in Lepidoptera,including Gln73 and Pro190 which are conserved to human NPC1 and NPC1L1.Gln73 is the key site to which NPC1b_N bounding hydroxyl of cholesterol.Human NPC1 inhibitors(ezetimibe and U18666A)had no inhibitory effect on the sterol absorption in the cotton bollworm,but promote the food intake.CRISPR-Cas9 can efficiently knock out NPC1 b gene in H.armigera and the insect cannot grow up to second instar if then lost this gene.The effect of different egg ages and sgRNA concentrations on the knockout efficiency were systematically evaluated.Knocking out efficiency was high generated if the eggs laid within 4 hours were used but the embryonic mortality was relatively high if the eggs laid within 2.5 hours were used.Knock out efficiency was similar between 50 ng/?l and 100 ng/?l sgRNA.In conclusion,NPC1 b is the key gene in absorbing dietary sterols in H.armigera.3)flotillin proteins have been reported to cooperate with NPC1L1 to transport cholesterol in human.Researches on flotillin involved in sterol absorption were rarely reported in insects.We found that flotillin proteins exist in all 23 genome-annotated insect species used in this study and these proteins can be divided into two groups,flotillin-1 and flotillin-2.By multiple sequence alignment analysis,the conserved sequences in flotillin were analyzed.These sequences can be used to quickly identify flotillin in insects.After flotillin-2 was knocked out,the efficiency of cholesterol absorption by cotton bollworm was decrease,suggesting that flotillin-2 may be involved in the intestinal absorption of sterols.This was the first analysis of flotillin protein evolution and sterol absorption in insects.4)Former research confirmed that SCP-X highly expressed in gut was involved in sterol transportation in insects including H.armigera.SCP2 domain is the function domain in SCP-X protein.Here,we identified the proteins containing SCP2 domain in the genomes of 10 species.The proteins containing SCP2 domain can be separated into four protein families: SCP2-X,SCP-2l,MFE2 and HSDL2.All insects possess one of the three proteins,MFE2,HSDL2 and SCP-X protein.But SCP-2l protein was only found in Lepidoptera and Diptera.Previous study indicated that one of SCP-2ls was highly expressed in the gut of A.aegypti and likely involved in sterol transportation.In contrast,SCP-2l was not gut-enriched in H.armigera.Consistent with previous report,SCP-X was highly expressed in the intestinal tract of the cotton bollworm and had four potential conserved sterol-binding sites,i.e.,F53,F89,F110,and Q131.This information provided the basis for the development of novel pest controlling strategies.5)Phytophagous insects cannot utilize phytosterols directly and need to convert phytosterols into cholesterol for growth.In this research,we used high-throughput sequencing technology to identify 378 DEGs involved in phytosterol metabolism.Eighty of them were annotated as lncRNAs.We then used the GO enrichment analysis and proteomic analysis to identify the candidate genes that were enriched in the gut.Among these genes,seven were selected for functional analysis.We used CRISPR/Cas9 to probe the function of these genes and found that metabolic efficiency of sitosterol,one major phytosterol,can be affected if these genes were knocked out.