| V-type ATPase(Vacuolar-type ATPase,V-ATPase)is an H~+transporter composed of multiple subunits,which is widely present in various endomembrane organelles of eukaryotes(such as endosomes and lysosomes)and plasma membrane of some specialized cells.V-ATPase pumps protons out of the cell or into the cytoplasm using the energy of ATP hydrolysis,which could maintain the normal p H value in the cell and are essential for growth and development of insects.This paper uses the important agricultural pest Locusta migratoria as the research object,and the molecular characteristics and biological functions of the V-ATPase genes are systematically studied.The main results are as follows:1.Analysis of the sequence and expression characteristics of Lm V-ATPase genesBy searching the locust transcriptome,we obtained all c DNA sequences of Lm V-ATPase genes.There was one c DNA sequence of Lm V-ATPase A,B,C,D,E,F,G,H,c,c",d and e,respectively,and two alternative splicing variants of Lm V-ATPase a(named Lm V-ATPase a1 and Lm V-ATPase a2).The identity of nucleotides of these two splicing variants was 95.5%,and the difference is only 30 bp.Gene structure analysis showed that these genes have a varying number of exons.The shortest and longest open reading frame length was 255 bp which encoded 84 amino acids,and 2562 bp which encoded 853 amino acids,respectively.Lm V-ATPase A has a N-terminalβ-barrel domain,Bulge domain,P-loop,hydrophobic loop,Walker A and Walker B motif.Lm V-ATPase B has a N-terminalβ-barrel domain and P-loop,but there is no Glycine-rich region in P-loop.Lm V-ATPase a,c,c"and e have 2-7 transmembrane regions.The expression characteristics of Lm V-ATPase genes in different tissues and developmental stages were studied by RT-q PCR,it showed that Lm V-ATPase A,B,C,D,E,F,G,H,c,c",d and e were highly expressed in malpighian tubule,while Lm V-ATPase a1 and a2 were highly expressed in ovary and testis,respectively.Besides,all genes were expressed every day from 3rd to 5th instar nymphs,among which Lm V-ATPase A,D,F,G,H,C,c’’,d and e were highly expressed in 3rd instar nymphs and Lm V-ATPase B,C,E and a1 were highly expressed in the end of 5th instar nymphs.Lm V-ATPase a2 was expressed in each day of5th instar nymphs.2.Functional analysis of Lm V-ATPase genes in L.migratoriaThe biological function of Lm V-ATPase genes in L.migratoria was studied by using RNAi technology.After injecting ds RNA of each Lm V-ATPase genes into 1-day old of3rd-instar nymphs,the transcription level of each gene was significantly reduced,and the mortality rate was 72.2%to 98.6%.After silencing Lm V-ATPase A,83.5%of nymphs could molt to 4th instar nymphs,with a curled wings,and eventually died.H&E staining showed that the structure of epidermis of wing injected with ds Lm V-ATPase A was loose and enlarged.Transmission electron microscopy(TEM)analysis revealed that the structure of epidermis of wing in control group was dense and evenly arranged.However,the structure of epidermis treated with ds Lm V-ATPase A was severely damaged.After silencing Lm V-ATPase a,the nymphs could develop normally to 4th instar nymphs,and then died continuously,with a high mortality rate of 98.6%.Meanwhile,when the nymphs developed to the first day of 5th instar,weight gains of nymphs decreased significantly.After dissecting their guts,it was found that there was almost no food residues in gut.H&E staining showed that goblet and columnar epithelial cells were arranged irregularly in ds Lm V-ATPase a injected nymphs,and the microvillar brush border seriously damaged.TEM analysis indicated that the microvillis of midgut were significantly shortened and a large number of vesicle-like structures accumulated in cell layer.After silencing Lm V-ATPase c,5.5%of nymphs died before molting,and 82.6%of nymphs died during molting.H&E staining revealed that although they could successfully separate the cuticle from the underlying epidermis known as apolysis,the new cuticle was significantly thinner,compared with the control.TEM analysis indicated that compared with the control,the new cuticle had a layered chitin structure,but its thickness was significantly thinner.After silencing other Lm V-ATPase genes,the locusts showed three phenotypes,died before molting of 3rd instar,died during molting of 3rd instar,and died after molting to 4th instar.3.Transcriptome analysis of wing and integument in L.migratoriaAfter silencing Lm V-ATPase A and Lm V-ATPase c,the transcriptome database of wing and integument of locusts was constructed,respectively.The differentially expressed genes were screened in the control and treatment groups by using locust transcriptome as a reference.The COG database was used to analyze the function of screened differentially expressed genes above.The transcriptome analysis showed that there are 146 up-regulated and 112 down-regulated differentially expressed genes in the transcriptome of wing.These differentially expressed genes were mainly involved in the secondary metabolites biosynthesis,transport and catabolism,DNA replication,recombination and repair,and energy production and conversion.Meanwhile,there are 256 up-regulated and 134down-regulated differentially expressed genes in the transcriptome of integument.Differentially expressed genes were mainly involved in the transport and metabolism of carbohydrates,amino acids,and lipids,as well as energy production and conversion.It is speculated that Lm V-ATPase genes participates in the development of locust wing and integument by regulating the transport,metabolism and energy production and conversion of various substances.However,the regulation mechanism of Lm V-ATPase genes needs to be further studied. |
