Genome-wide Identification Of Genes Encoding ATP Synthase Subunits And Their Response To Juvenile Hormone In The Silkworm

ATP is an important energy currency of organism which mainly synthesized by the oxidative phosphorylation in mitochondria.The Oxphos system,an exothermic reaction of hydrogen and oxygen combining into H2 O,which consist of the electron transport chain,is located in the inner mitochondrial membrane.Reduced NADH and FADH2 produced in TCA cycle can be further oxidized through a series of redox reactions in the mitochondrial inner membrane,and release free energy supplying for ATP syntheses pumping H+ across the mitochondrial inner membrane and catalyzing phosphorylation of ADP to ATP.ATP synthase is an enzyme complex,mainly composed of three main units:FO,F1 and the outer handle connecting the two parts.As the conformational coupling hypothesis says,when proton flow going though FO unit of ATP synthase,conformation of ? subunit changes to open situation and release ATP from the enzyme.Juvenile hormone(JH),synthesized in the corpora allata(CA),is one of the most important endocrine hormone in silkworm which regulates the growth and development of silkworm together with 20-hydroxyecdysone(20E).The titer of JH has a peak in the early of every larval instar to promote physiological metabolism and keep development of larva;20E is responsible for the initiation of larval molting and metamorphosis molting into pupation and moth.JH and ATP synthase are both involved in the regulation of growth and development,while the connection between them is not clear.Silkworm is an important economic insect,also used as a model organism of insect genetic and physiological research.We compare and analyse the genome and transcriptome data,identify genes encoding ATP synthese subunit in the silkworm genome and establish the spatio-temporal expression pattern of these genes.We further research the influence of JH on expression of genes encoding ATP synthase subunits and ATP synthesis,which is derived from cells,tissues,and individual level.The main research results are as follows: 1.Identification of genes encoding ATP synthase subunits of the silkwormGenes encoding ATP synthase subunits of silkworm are identificated by homology BLAST in two silkworm genome databases including SilkDB(http://www.silkdb.org/ silkdb/)and KAIKObase(http://sgp.dna.affrc.go.jp/ KAIKObase/)with a query of ATP synthase amino acid sequences of drosophila.Based on the similarity of sequence and conservative of functional domains,13 genes encoding ATP synthase subunits in silkworm nuclear genome are found.Further bioinformatics analysis suggests that these genes have distinct structure domain,different numbers of exons varying from 1(ATPB)to 10(ATPA)and lengths of amino acid sequence alter from 61(ATPE)to 553(ATPA),while all of them can be located in the specific chromosome except ATPE.Comparative genomics analysis shows that all genes have single copy in silkworm,fly,monarch,red flour beetle and bee,but AT91 has three copies in mice and human.Phylogenetic analysis clusters these homologous genes with same single copy in different insects to a cluster,while them in mice and human to another cluster.Interestingly,each copy of AT91 in mice and human are clustered before clustering with other copy branches,implying that the multicopy of AT91 appears before species differentiation in humans and mice.2.The spatio-temporal expressions of ATP synthase subunit genes in silkwormWe analysis the spatio-temporal expression patterns of ATP synthase subunit genes.Firstly,we found that the expression patterns of ATP synthase subunits are similar,show that high expression in midgut and Malpighian tubules,two metabolism tissues and head and that less expression of epidermis,a metabolism tissue,through genome-wide microarray data of male and female silkworm larval tissues at the third day of fifth instar.Secondly,we found that the ATP synthese genes are expressed at the third day of fifth instar,and more highly at the third day after pupation by analysising transcriptome data of silkworm fat body during metamorphosis development process.Finally,the analysis of genome-wide microarray data of silkworm larva from the forth day of fifth instar to adult indicates that ATP synthase subunit genes are highly expressed during the fifth instar of larva and the stage after pupation.Comprehensive analysis of the results above shows that the genes encoding silkworm ATP synthase subunits are highly expressed during the larvae growth stage and the development stage from pupae to adult and mainly in the metabolism tissues during the larval stage,which is consistent with the specific trend of active metabolic activity space and time of silkworm growth and metabolic.3.JH has an effect on ATPO expression and ATP synthesisWe explore the effects on the genes expression of ATP synthese subunits of JH.With JH analogue(JHA)treatment of silkworm(BmE cells)and in vitro culture of silkworm epidermal tissue,we analyze the expression of silkworm ATP synthase subunit genes by quantitative RT-PCR.The results show that JHA treatment leads to a significant increase of ATPO expression,and the expression level of ATPO is also up-regulated significantly in the carcass by injecting with JHA.Further quantitative RT-PCR results show that ATPO is mainly expressed at the early stage of the forth instar and less at the fifth,which is consistant with the change trend of JH titer in silkworm haemolymph,suggesting that JH regulates the transcription of ATPO positively.We further analyze the effects on silkworm ATP synthesis of JHA treatment.Results show that both BmE cell line and silkworm individuals,JHA treatment can promote the synthesis of ATP.Overexpression of Kr-h1,the signaling transduction molecule of JH pathway,in BmE cell line leads to a significant increase of ATPO expression and ATP content.RNAi treatment of ATPO in BmN4-SID1 cell line,which is from silkworm ovary,can lead to a remarkable decrease in ATP content.In conclusion,we hypothesize that JH accelerates the synthesis of ATP by promoting ATPO gene expression.