Functional Analysis Of Polyadenylate Binding Proteins During The Molting Process Of Locusta Migratoria

There are about one million species of insects,accounting for the highest proportion of all animal kingdoms,including Arthropoda,which has a wide range of terrestrial habitats.However,insects do not have the skeletal system of higher animals,and their epidermis acts as skin and bone.Due to the lack of elasticity of the epidermis,when the larva grows and develops to a certain stage,it will be limited by the inelastic epidermis.At this time,in order to continue to grow,it must break through the limitation and grow a new epidermis,which is called molting.Insect molting is an important process for the growth and development of insects.The whole molting process involves multiple complex physiological and biochemical processes such as cortical dissolution,digestion and absorption of old epidermis,and formation of new epidermis.During this rapid molting process,the formation of new epidermis involves the production of a large number of m RNAs,and with the degradation of the old epidermis,there is also a large amount of attenuation of the original m RNAs.Finally,m RNAs can reach a highly stable state and carry out complex physiological and biochemical processes in an orderly manner,which are closely related to Polyadenylate-binding protein(PABP).A review of relevant literature shows that PABP binds to the Poly A tail of eukaryotes and plays an important role in m RNA stability,translation and m RNA decay.However,the stability mechanism of PABP on molting key genes during insect molting is still unclear.In this paper,Locusta migratoria was taken as the research object to deeply analyze the biological function of polyadenylate binding protein during its molting process.The specific functional differences between cytoplasmic PABP(LmPABP1)and nuclear PABP(LmPABP2)genes and the mechanism of action on the m RNA stability of key molting genes were compared and analyzed.The main research results are as follows:1.Sequence acquisition and expression analysis of LmPABPs gene in L.migratoriaBased on the transcriptome database of L.migratoria,the c DNA sequences of two LmPABPs genes were selected and named as LmPABP1 and LmPABP2 respectively.Bioinformatics analysis showed that: the Open Reading Frame(ORF)length of LmPABP1 was 1887 bp,encoding 628 amino acids.Amino acid sequence analysis revealed that LmPABP1 contained four functional domains of conserved RNA recognition motifs(RRMS).The C terminal contains a Poly A domain(PABC);The ORF length of LmPABP2 was 690 bp,encoding 229 amino acids.The amino acid sequence analysis showed that LmPABP2 contained only one RRM functional domain,lacking the C terminal domain.Phylogenetic tree analysis showed that the two selected polyadenylate binding proteins were divided into two categories,among which LmPABP1 belonged to the first category and was classified as cytoplasmic polyadenylate binding protein(PABPC/PABP1).LmPABP2 belongs to the second group and is classified as nuclear polyadenylate binding protein(PABPN/PABP2).The expression of LmPABP1 gene in tissues and developmental expression pattern showed that LmPABP1 was expressed in each instar,and the expression was higher in the fifth instar.Tissue expression showed that it was highly expressed in the epidermis,foot and testis of the fifth instar.LmPABP2 is also expressed in each instar,and the expression of LmPABP2 increases first and then decreases in each instar.The tissues expression shows that LmPABP2 is only highly expressed in the testis.2.Functional study of LmPABPsThe function of LmPABP1 and LmPABP2 genes in the epidermal development of nymph-nymph stage and nymph-adult stage was investigated by RNA interference(RNAi)technique.The results showed that two different phenotypes appeared when the expression of LmPABP1 and LmPABP2 genes were significantly inhibited.The total mortality rate of the fourth instar nymph reached 100% after injection of 8 μg ds LmPABP1.After injection of 10 μg ds LmPABP1 into the fifth instar nymphs,all of them were in the state of death before molting and not cracking at the ridge line.However,after injection of ds LmPABP2 at age 4 or 5,death and successful survival phenotypes were observed before,during,and after molting.H&E staining results showed that epidermal developmental delay was observed at day 7 during the 5th age period(N5D7)in the experimental group after injection of ds LmPABP1 and ds LmPABP2,and no degradation of the old epidermis and no formation of the new epidermis compared with the control group injected with ds GFP.3.Differential expression gene analysis and m RNA stability experiment based on RNAiIn order to further elucidate the differences between LmPABP1 and LmPABP2 in insect molting process and explore the mechanism of LmPABPs(LmPABP1 and LmPABP2)in the stability of m RNA of key genes in insect molting process,First,transcriptome sequencing was performed on the epidermis of nymphs in the group injected with ds RNA and the control group injected with ds GFP respectively.The down-regulated differentially expressed genes were screened and analyzed based on RT-q PCR technology.Then,the m RNA stability of down-regulated differentially expressed genes was tested using Act D(Actinomycin D).The stabilization mechanism of LmPABPs was discussed,and the results showed that:(1)The number of differentially expressed genes was counted: in the experimental group injected with ds LmPABP1 and the control group injected with ds GFP,A total of 33 differential genes were identified,including 17 up-regulated genes and 16down-regulated genes.And in the control group and the group injected with ds LmPABP2,a total of 11 differential genes were identified,including 5 up-regulated genes and 6 downregulated genes.These differentially expressed genes mainly included chitin metabolism genes and some unknown functional genes.(2)Differential expression gene enrichment analysis showed that,in the GO enrichment classification,it is mainly concentrated in biological processes such as cellular process,stimulus response,detoxification and metabolism.In the KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis,the genomes were mainly distributed in m RNA monitoring pathways,RNA degradation pathways,RNA transport pathways,peroxidase pathways,drug metabolis-other enzymes pathways,etc.They differ from COG classification by Cluster of Orthologous Groups of proteins,which are mainly distributed in lipid transport and metabolism,translation,ribosome structure,biosynthesis and catabolic pathways.(3)RTq PCR based verification of down-regulated differentially expressed genes found: Among them,the genes related to chitin metabolism,Lm Cht5-1,Lm Cht10,Lm UAP1,and the unknown functional genes LOCMI09926,LOCMI15653,LOCMI00852,were significantly down-regulated in the experimental group injected with ds LmPABP1,and the m RNA stability experiment showed that,the m RNA degradation rate of chitin degradation-related genes,Lm Cht5-1 and Lm Cht10,was accelerated,and the instability was enhanced.The m RNA of Lm UAP1,which is related to chitin synthesis,and LOCMI09926,LOCMI15653 and LOCMI00852,which are unknown functional genes,showed accumulation and increased stability.In the control group and the experimental group that interfered with LmPABP2 expression,the unknown functional genes LOCMI15653 and LOCMI00852 were also significantly down-regulated,and the m RNA stability experiment showed that their m RNA also showed accumulation and enhanced stability.(4)In order to further explore the unknown functional genes LOCMI09926,LOCMI15653,LOCMI00852,bioinformatics analysis and biological function research based on RNAi were carried out.The results showed that the protein sequence encoded by LOCMI15653 contained a signal peptide and a conserved OS-D motif related to odor-binding proteins.The amino acid sequence analysis results showed that the amino acid sequence of this gene was very similar to that of chemoreceptor protein of Oedaleus asisticus,and the evolutionary tree shows the two genes clustered into one branch,therefore,LOCMI15653 was speculated to be the gene encoding chemoreceptor protein of L.migratoria.The functional annotation of LOCMI09926 encoded protein showed that it was an animal heme peroxidase.Amino acid sequence alignment and phylogenetic tree analysis indicated that it was conserved among species,and it was speculated that it is presumed to be the gene encoding heme peroxidase of L.migratoria.Based on RNAi technology,compared with the control group injected with ds GFP,no visible phenotype was observed in the experimental group injected with ds LOCMI15653,ds LOCMI00852 and ds LOCMI09926.Based on RNAi technology,this paper studied the biological function of LmPABPs in the molting process of L.migratoria,analyzed and compared the functional differences between LmPABP1 and LmPABP2 through transcriptome sequencing,and further tested the m RNAs stability of down-regulated genes by detecting differential expression.To elucidate the stability mechanism of LmPABPs on key genes during molting process,which provides a new target and a new idea for pest control technology based on molting process.