The Sugar Content Change And The Expression Analysis Of Trehalose Phosphate Synthase(ApTPS)Gene In Chinese Oak Silksorm,Antheraea Pernyi During The Pupae Diapause Termination

The Chinese oak silkworm(Antheraea pernyi)belongs to lepidoptera bombycidae Antheraea spinning insect accounts for important state in the national economy in China,which is a important unique economic insect in China.Antheraea pernyi is a holometabolic insect,and is of the pupa diapause insect.Antheraea pernyi experiences four differently physiological stages of the egg,the larva,the pupa and the adult in their life.The study of the tussah pupa diapause termination can turn "the twice stocking of the univoltine tussah" into realization.Here,we selected Shen Huang 1 of Insect Resource Engineering Research Center of Liaoning Provice in the Shenyang Agricultural University and Yu Da 1 of the Henan Sericultural Institute as basic material.The study contained the analysis of the tussah feather level during the period of diapause termination,the detection of sugar content,the cloning of trehalose phosphate synthase gene(ApTPS),the bioinformatics analysis of ApTPS gene,the expression profile of ApTPS gene in different tissues,the changes of ApTPS gene expression in the different stages,and the analysis of the prokaryotic expression of ApTPS.The results are as follows:1.The study of diapause termination was conducted by Antheraea pernyi Yu Da 1 and Shen Huang 1 explored in the various light combinations.Meanwhile,the body fat and blood lymphatic were taken regularly.Anthrone colorimetry was used to detect trehalose and glycogen content.The results showed that 17 h with 400 Lux was found to be best for Yu Da 1 diapause termination with the feather peak time and the correction eclosion rate of 80%.As for Shen Huang 1,17 h with 260 Lux was to achieve the highest calibration eclosion rate of 89.9%;17 h with 400 Lux was to reach the easiest feather perk.The average pupal eclosion rate of Yu Da 1 was relatively lower 9.1%and feather delay 19.16 days than Shen Huang 1.2.Trehalose content and glycogen content were detected during the period of diapause termination.The result showed Yu Da 1 and Shen Huang 1 trehalose content had no obvious change regulation,under the natural light processing,Yu Da 1 glycogen content had no obvious change regulation,under 17 h light processing,Yu Da 1 glycogen content showed a trend of decline after rising first.However,in 17 h light and natural light illumination treatment,Shen Huang 1 glycogen content performanced continuously decline trend.3.ApTPS gene was obtained by the modern molecular biology technology.The results showed that ApTPS gene(GenBank accession NO.KU977454)has an open reading frame(ORF)of 2 487 bp,encoding 828 amino acid residues by bioinformatics.The predicted protein is estimated to be 93.19 kDa with an isoelectric point(pI)about 4.61.There is no signal peptide and transmembrane region.Subcellular localization analysis indicates that ApTPS is targeted to the cytoplasm.Homology searching shows that ApTPS has two conserved domains:TPS(22-497)and TPP(532-772).4.The expression profile of ApTPS gene is conducted by semi-quantitative PCR in the different tissue,which revealed ApTPS gene mainly expressed in the fat body of A.pernyi larvae.Real-time PCR technology is used to detect the expression of ApTPS gene in the process of diapause termination,which were induced the expression level of ApTPS gene in haemolymph and fat body both upgraded significantly(P<0.05),and that the change of the gene in haemolymph was later than that in fat body.5.We designed primers,constructed prokaryotic expression vector for ApTPS gene with plasmid pET-28a and successed to express the purpose protein in BL21(DE3).Then we carried on the protein purification,the manufacture of antibody and Western Blot identification,etc.The results showed the construction of prokaryotic expression vector was successed.The rugelation of ApTPS in the fat body was similar with the reslt the real-time quantitative conclusion basically in the process of diapause termination by Western Blot analysis,which showed a trend of decline after rising first.