Functional Analysis Of LmFHL2 In The Molting Process Of Locusta Migratoria

Insects are the oldest and most widespread terrestrial taxa on the Earth,which has a wide distribution and strong adaptability.The important reason for their superior adaptability is the integument of the insect.The insect integument is composed of basement membrane,epidermis and the cuticle that secreted by epidermal cells.Different to human skin,the integument does not grow with the insect development.When the body of the insect grows,the original cuticle cannot adapt to the growth of the body.The insect will carry out cyclical moulting,and the new cuticle will replace it.Moulting will be carried out at every instar until it grows into an adult.Life activities of the organisms are inseparable from the three major energy substances,protein,lipid and glucose.The insect cuticle is a metabolic reservoir that carries out the metabolisms of amino acids,lipids and glucoses constantly.Studying and analyzing the molecular characteristics and functions of important genes involved in the molting period,with a vigorous metabolism,and elucidating their mechanism of action will provide certain ideas and methods for pest control.Locusta migratoria was selected as the research object in this thesis.By the analysis of the transcriptome database of the integument of the L.migratoria,the lethal Four and a half LIM domain 2(FHL2)gene was screened.Its molecular characteristics and expression patterns were analyzed,and the effect of LmFHL2 on the growth and development of the L.migratoria molting process was explored based on RNA interference(RNAi),and the results are shown as follows:1.Analysis of molecular characteristics and expression patterns of LmFHLs gene in L.migratoriaBased on the gene clusters of six time nodes in the cuticle transcriptome of L.migratoria,the lethal gene Lm12533 was screened through RNAi,and the protein name was FHLs.When the LmFHLs gene sequence,exon and protein structure were analyzed,it was found that it has splice,that is,LmFHL2 and LmFHL3.The sequence of LmFHL2 was completely consistent with the second half of the LmFHL3 sequence.LmFHL3 had10 exons and LmFHL2 had 3 exons.Similarly,the structure of FHLs protein in other species was analyzed and found that such sheared forms also exist in other species,but Homo sapiens and Mus musculus in mammals do not have such sheared forms unlike these species.At the same time,a phylogenetic tree was constructed,and LmFHL2 and LmFHL3 of the L.migratoria were combined with Nlu FHL2 and Nlu PRICKLE3 of Nilaparvata lugens.Most insects have FHL2 and FHL3,except for the Hymenoptera only have FHL2.The expression analysis of different tissues showed that LmFHL2 was highly expressed in the integument of L.migratoria,and the expression was high before and after molting,and the expression level was higher in the adults than that in the nymphs.While,LmFHL3 expressed not high in the integument,and the expression trend is similar to LmFHL2 throughout the period,but both are low.2.Functional analysis of LmFHLs gene in L.migratoriaRNAi technology was used to study the biological functions of LmFHL2 and LmFHL3.Fifth instar nymphs of L.migratoria were injected with ds LmFHL3 and ds GFP.By observing the phenotype,it was found that it only led to the curling of the adult wings after molting and the phenotypic effect was not obvious after injection the ds LmFHL3.At the same time,ds LmFHL2 and ds GFP were injected into the first day of the fourth instar and the first day of the fifth instar,and it was found that there was no abnormality in the molting to the fifth instar,while the L.migratoria died before it molt from the fifth instar to the adult,and there was also a lethal phenotype during the molting process,and the wings of all adults were significantly abnormal after molting.In view of the fact that the phenotypic effect of L.migratoria after ds LmFHL3 is not obvious,LmFHL2 was studied in a follow-up concentration.Subsequently,dynamic analysis was performed using paraffin section stained with hematoxylin and eosin(H&E).The results showed that the new cuticle formation of L.migratoria was delayed after injection of ds LmFHL2.The cuticle of L.migratoria that molt to death is abnormal development,and the fat body under the epidermis is more scattered.By measurement and weighing of L.migratoria bodies before and after molting,it was showed that ds LmFHL2-injected L.migratoria had lower body weight,smaller individuals,and smaller wing areas after molting compared with ds GFP injection.3.Combined analysis of RNAi transcriptome and metabolomeThe integument of L.migratoria after injection of ds LmFHL2 and ds GFP was sampled for transcriptome sequencing.The results showed that they had a total of 132 differentially expressed genes,with 22 up-regulated genes and 110 down-regulated genes.The down-regulation genes were verified by RT-q PCR,and it was found that all of them were down-regulated,and the down-regulation trend was positively correlated with the gene expression level.Analysis of differential enrichment pathways found that most of these differentially expressed genes were related to glucose,lipid,amino acid and nucleotide metabolism.By sampling the integument of the L.migratoria death during molting after ds LmFHL2 injection,and the integument of ds GFP injection in the corresponding period to detected for metabolome detection,it was found that there were1526 differential metabolites,among which,1020 downregulated metabolites,and 506up-regulated metabolites.After the important metabolites and the important pathways analyzing,it was found that they were consistent with transcriptome results,which indicated these metabolites mainly involved in the metabolism of glucose,lipids,amino acids and nucleotides.Subsequently,the total content of amino acids,triglycerides and glucoses was determined,and the content was significantly reduced compared with the control.In summary,this thesis analyzes the molecular characteristics of LmFHL2 gene screened during the molting process of L.migratoria,studies the biological function of LmFHL2 gene by RNAi technology,and analysis of the transcriptome and metabolome.It is found that LmFHL2 gene affects the metabolism of glucose,lipid,amino acid and nucleotide,and lead to the shortage of the three major energy substances,glucose,lipid and protein,which are necessary for life activities,thereby the important substances during the molting process is difficult to synthesis.Together with the power is insufficient,it leads to molt difficulty and died eventually.Even some locusts can molt to adult,they also abnormal development due to inadequate nutrition.The above research results enrich the research of related genes in the molting process of L.migratoria,and provide certain ideas and methods for the prevention and control of L.migratoria.