| Galeruca daurica(Joannis)is a new pest that has caused serious harm to ecological environment and animal husbandry in Inner Mongolia grasslands in recent years,feeding as larvae and adults on the foliage of Allium plants.The insect is a poikilotherm,and temperature has an important influence on its growth,development,survival and reproduction.Trehalose,as the blood sugar of insects,not only conducts energy metabolism,but also plays an important role in resisting the high and low temperature stress,affecting the normal development and resilience of insects.Trehalose-6-phosphate synthase(TPS)and Trealose-6-phosphate phosphatase(TPP)are two of key enzymes in the trehalose synthesis pathway.In this study,based on the transcriptome data of the Galeruca daurica larvae,the TPS and TPP genes were identified,cloned,and characterisitized.Their expression in response to temperature stress analyzied,and the prokaryotic expression vector for TPP gene was successfully built.This research aims to investigate these their roles in resistance to high temperature and low temperature environments,and to provide a necessary foundation for further functional research on the molecular mechansims of stress resistance in Galeruca daurica.The main results are as follows:1.Cloning and biological information analysis of TPS and TPP genes in Galeruca dauricaBy analyzing the transcriptomic data,cDNA fragments of TPS and TPP genes were obtained,and the specific primers of two genes were designed.The full-length cDNA sequences of TPS and TPP genes in Galeruca daurica were cloned by RACE method and named GdTPS(GenBanka accession No: KY460114)and GdTPP(GenBanka accession No: MG431210),respectively.GdTPS is 2706 bp in length with an open reading frame(ORF)of 2496 bp,encoding a protein of 831 amino acids with the predicted molecular weight of 94.05 kD and pI of 6.82.The encoded protein contains three potential N-glycosylation sites,without signal peptide and transmembrane domain.GdTPS has two conserved domains,shares higher amino acid sequence identity with TPSs of other insect species,and has the highest amino acid sequence identity(88%)with TPS from Leptinotarsa decemlineata.GdTPP is 1372 bp in length with an open reading frame(ORF)of 864 bp,encoding a protein of 287 amino acids with the predicted molecular weight of32.32 kD and pI of 6.19.The encoded protein contains two potential N-glycosylation sites.The subcellular localization prediction shows that the encoded protein is located in the cytoplasm without signal peptide and transmembrane domain.GdTPP has one conserved domains,shares higher amino acid sequence identity with TPPs of other insect species,and has the highest amino acid sequence identity(68%)with TPP from Leptinotarsa decemlineata.2.Expression analysis of the TPS and TPP genes in response to temperature stress in Galeruca dauricaReal-time quantitative PCR results showed that when the stress temperature was lower than 25?(the control),the expression level of GdTPS and GdTPP increased as the temperature decreased,and reached the peak at-10 ? with 2.22-fold and 1.86-fold compared with the control at 25?,respectively.However,when the stress temperature was higher than 25?,its expression level increased as the temperature increased,and reached the peak at 40 ? with 1.58-fold and 1.68-fold compared with the control at 25 ?,respectively.Therefore,short-term cold stress has a greater influence on the expression of TPS and TPP genes than short-term heat stress in Galeruca daurica.3.Prokaryotic expression of GdTPPTo explore the function of GdTPP,prokaryotic expression system was used to express GdTPP.By inserting the GdTPP gene into prokaryotic expression vector pET-28a(+)to construct recombinant plasmid pET-GdTPP,the recombinant plasmid was transformed into E.coli BL21(DE3)to express.The result of SDS-PAGE electrophoresis indicated that GdTPP gene was expressed in E.coli,and the molecular mass of recombinant protein is about 32.3 kD in the agreement to the speculated size. |
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