The Expression Of Eclosion Hormone-related Genes And The Research Of Trehalase Activity Of Antheraea Pernyi

Diapause is an important physiological for insects,mainly characterized by the stagnation of growth and development and the reduction of physiological activities so that insects can respond to unfavorable conditions to maintain the continuation of species.The A.pernyi is a silkworm of the genus Tussah,a genus Lepidoptera with pupae diapause.It is an economic insect with Chinese characteristics.A.pernyi pupae entering diapause and the diapause termination is an important part of the protection and production of A.pernyi seed,and the research on its hormone regulation mechanism is the basis for artificially controlling the eclosion of A.pernyi pupae in different voltinism areas of A.pernyi.So,the research on the regulation mechanism of A.pernyi diapause and diapause termination is important for the production and clarification mechanism of A.perny.The "Shenhuang No.2" conserved by the Tussah Research Institute of Shenyang Agricultural University and the "Yuda No.1" conserved by the Henan Institute of Sericulture which were selected as materials.The molecular biology method was used to study the mechanism in the hormonal regulation of eclosion hormone(EH)、ecdysis triggering hormone(ETH)、 ecdysis triggering hormone receptor(ETHR)genes and in sugar metabolism regulation of the trehalase(TREH)gene in A.pernyi pupae during diapause and diapause termination.The result is as follows:1.The research of eclosion hormone cloning and expression of A.pernyiUnigene sequence annotated as eclosion hormone gene were screened according to the transcriptome database of A.pernyi midgut established in our laboratory,and primers were designed using Primer premier 5.0 software.The eclosion hormone gene was cloned from A.pernyi named ApEH.The open reading frames(ORF)of gene is 267 bp in length,encoding 88 amino acids.Semi-quantitative RT-PCR showed that ApEH was highly expressed in the brain in diapause and developmental stages of A.pernyi pupae and EH was highly expressed in the spermary/ovary during diapause and in the fat body during development and weakly expressed in other tissues.qRT-PCR detected that after long-light treatment of diapause the expression level of EH was maintained at a low expression level between 0 and 21 d,and began to increase at 21 d and reached the highest level at 35 d and then began to decline,and reached the lowest level at 49 d.Combined with the results of respiration intensity measurement,long light accelerated the diapause termination of A.pernyi and promoted its development.The results showed that the diapause pupae of A.pernyi did not start the eclosion process within 20 days after long light treatment and the eclosion process started after 21 days.2.The research of ecdysis triggering hormone and ecdysis triggering hormone receptor genes cloning and expression of A.pernyiPrimers were designed according to the transcriptome database of A.pernyi midgut using Primer premier 5.0 software.The ecdysis triggering hormone and ecdysis triggering hormone receptor genes were cloned from A.pernyi named ApETH and ApETHR-A respectively.The open reading frames(ORF)of two genes are 264 bp and 1 653 bp in length,encoding 87 and 550 amino acids respectively.Semi-quantitative RT-PCR showed that ApETHR-A were highly expressed in the brain in diapause and developmental stages of A.pernyi pupae.ETH was highly expressed in the fat body of diapause and in spermary/ovary during development and not expressed in other tissues;ETHR-A was highly expressed in spermary/ovary during diapause and in fat body during development and weakly expressed in other tissues.qRT-PCR detected that ETH was highly expressed when the larva appeared just pupae and integument was not hardened and blackened(commonly known as "Shenxian pupae").Then the expression level of ETH began to decline and reached the lowest level at 21 d and then increased and reached the highest level at 49 d.The relative expression level of ETHR-A was higher at 0 d,then decreased slowly and reached the lowest level at 35 d and reached the highest level at 49 d.The results suggested that the expression trends of eclosion hormone,dehulling promoter hormone、ecdysis triggering hormone and ecdysis triggering hormone receptor genes were basically same variation in the process of A.pernyi pupae near eclosion suggesting that the expression responses of the three genes played an important role in A.pernyi pupae near eclosion.3.The research of trehalase genes cloning and expression of A.pernyiPrimers were designed according to the transcriptome database of A.pernyi midgut using Primer premier 5.0 software.Three trehalase genes were cloned from A.pernyi,named ApTreh1 A,ApTreh1B and ApTreh2 and deposited in GenBank under accession numbers KU977455,KU977456 and KU977457,respectively.Their open reading frames(ORFs)are 1 797,1 635 and 1 932 bp in length,encoding 598,544 and 643 amino acids,respectively.Homologous sequence alignment and phylogenetic analysis indicated that ApTreh1 A and ApTreh1 B are soluble trehalases(TrehS),and ApTreh2 is a membrane-bound trehalase(TrehM).Tissue-specific mRNA expression profiling using semi-quantitative RT-PCR showed that ApTreh2 had a wider distribution and higher expression level than ApTreh1.The qPCR results indicated that the expression levels of ApTreh1 A and ApTreh1 B in fat body at 21 d after exposure to long photoperiod were up-regulated and significantly higher than that of the control group(12L:12D)(2-and 4.7-fold,respectively),while down-regulated at 28 and 35 d,and then up-regulated again at 42 d.The expression levels of ApTreh2 were up-regulated gradually,and reached the maximum(2.7-fold as high as that of the control group)at 28 d.At 42 d after exposure to long photoperiod,there was another expression peak(2.3-fold as high as that of the control group),and then down-regulated gradually.4.Study on the enzyme activities of trehalases and the change of trehalose content in A.pernyi pupae during diapause and diapause terminationThe trehalase activity in the fat body was detected using 3,5-dinitrosalicylic acid method and the trehalose content in the haemolymph was measured using antrone chromametry method.Trehalase activities in fat body increased gradually,reached the peak(about 18.5 U)at 21 d after exposure to long photoperiod,while declined to the lowest level(11.2 U)at 35 d,and increased slightly at 42 d,showing the same variation trend as the gene expression.The trehalose content in the haemolymph increased under long photoperiod and reached the maximum at 21 d,and was higher than that of the control during the whole developmental stage.The results suggest that the expression of trehalase genes shows the same variation trend as the trehalase activities in the fat body and the trehalose content in the haemolymph in A.pernyi,suggesting that the expression response of trehalase genes plays a significant role in pupal diapause and diapause termination of A.pernyi.