The Cloning And Expression Analysis Of Genes Related To Trehalose Metabolism In Gampsocleis Gratiosa

Trehalose [?-D-glucopyranosyl-(1,1)-?-D-glucopyranoside],a nonreducing disaccharide formed by an ?1–?1 bond between two glucose molecules,which plays a crucial role by stabilizing dehydratase,protecting membranes and proteins in the adaptation to environmental extremes including heat,freezing,anoxia,desiccation,dehydration,and nutrient deficiency.Our findings may provide theoretical basis for proving stress-resist ability of insects.Based on the Gampsocleis gratiosa transcriptome sequencing data set,the full cDNA of the G.gratiosa TPS and Tre gene were cloned.This study aims to ascertain the effects of rapid vs gradual change in temperature on trehalose-6-phosphate synthase(TPS)gene expression level,and trehalose,and total sugar,content,in the haemolymph,as well as the tissue-specific expressions of trehalase(Tre)genes in G.gratiosa.Rapid and gradual change in temperature treatments were applied to G.gratiosa.The body temperature of G.gratiosa was measured with a thermocouple thermometer,and the environmental temperature closest to body temperature was regarded as the optimal temperature.TPS and Tre genes expression were detected using real-time fluorescent quantitative PCR.Sulfuric acid-anthrone colorimetry was employed to measure trehalose,and total sugar,content(the control was the normal content at 25?).The full-length cDNAs of a TPS gene,a soluble trehalase gene(Tre1)and a membrane-bound-like trehalase gene(Tre2-like)were cloned in G.gratiosa.TPS was designated as GgTPS(GenBank accession no.KU578006).The full-length cDNA of GgTPS is 3 225 bp with 5?-and 3?-untranslated region of 153 bp and 642 bp,respectively.It contains an open reading frame of 2 430 nucleotides encoding a protein of 809 amino acids residues with a predicted molecular mass of approximately 91.35 ku and isoelectric point of 6.28.The two GgTre genes c DNA were cloned and designated as GgTre1,GgTre2-like,respectively.Putative GgTre1 cDNA has three isoforms(GenBank accession number KY400001~KY400003)with the lengths of 2 107,2 021,1 914 bp,respectively.All of the three isoforms of GgTre1 share the same 5?-untranslated regions(5'-UTRs)with 33 bp,but differ in the length of the 3?-UTRs with 322,248,and 129 bp,respectively.The complete ORF sequence of isoform 1/3 is 1 752 bp,which encodes a 583 amino acids protein,with a calculated molecular weight of 67.88 ku and an isoelectric point of 6.59.The transcript isoform 2 with an ORF of 1 740 bp,encodes a 579 amino acids protein with a calculated molecular weight of 67.29 ku,and an isoelectric point of 6.34.Three isoforms(Gen Bank accession number KY400004~KY400006)with the lengths of 2 491 2 460,2 381 bp,respectively for GgTre2-like cDNA.Three GgTre2-like isoforms share the same 5?-UTRs(284 bp)and coding regions,but differ in the length of the 3?-UTRs with 398,367,285 bp,respectively.The complete ORF sequence is 1 809 bp,which encodes a 602 amino acids protein,with a calculated molecular weight of 67.88 ku and an isoelectric point of 6.59.During the rapid cooling phase,TPS expression first increased,then decreased,peaking at 15?,which was in marked contrast to that measured at 25?.Trehalose content remained constant level before increasing to a high level at 0?.Total sugar content increased gradually,reaching a maximal level at 0?.During the rapid heating stage,TPS had a down-up-down expression profile,peaking at 40?.Trehalose content was significantly lower at 45?.Total sugar content was markedly different at 40? and 45?.During the gradual cooling stage,TPS expression first increased,then decreased,and was highly expressed at 15?and 20?.Trehalose content first decreased,then increased,and was significantly higher at 0?.Total sugar content was markedly different at 0?,10? and 20? to that measured at 25?.During gradual heating,TPS expression had no marked difference to that measured at 25?.Trehalose content was significantly lower at 40? than 25?.Total sugar content first increased,then declined,and was significantly higher at 40? and 45? than at 25?.Comparing rapid vs gradual change in temperature,TPS expression differed significantly at 15? and 40?,and trehalose content was markedly different at 40? and 45?.Total sugar content was markedly different at 10? and 20?,and body temperature was also markedly different at 30?.GgTre1 was highly expressed in the ovary and accessory gland.GgTre2-like was mainly expressed in ovary.GgTre2-like was significantly higher expressed in the muscle and Malpighian tubules than in other tissues in male carcas.The results revealed that temperature acclimatisation improves the ability of insects to resist stress.The TPS expression levels and trehalose content of insects are closely related to ambient temperature,as well as the duration of temperature acclimatisation.Patterns of TPS expression and trehalose accumulation in G.gratiosa kept at low temperature differ from those at high temperature.GgTre1 and GgTre2-like gene has progressive lengthening of 3'-untranslated regions,which resulted from alternative polyadenylation(APA).GgTre1 expression level varies considerably among different tissues compared to GgTre2-like gene,which presented a relatively balanced gene expression level.GgTre1 and GgTre2-like tissue distribution differes.