Molecular cloning and prokaryotic expression of the trehalose-6-phosphate phosphatase gene GdTPP in Galeruca daurica during temperature stress
Author of the article:LU Biao1** ZHOU Xiao-Rong1 PANG Bao-Ping1*** DONG Rui-Wen2 NABUQIYA2 NARENMANDUHU2
Author's Workplace：(1. Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010020, China; 2. Siziwangqi Grassland Station, Siziwangqi 011800, China
Key Words：Galeruca daurica; trealose-6-phosphate phosphatase; gene cloning; temperature stress; prokaryotic expression; expression profiling
Abstract：
[Objectives] To clone the Trealose-6-phosphate phosphatase (TPP), a key enzyme in the trehalose synthesis pathway, in Galeruca daurica, quantify its expression during temperature stress, and lay a foundation for further investigation on the role of this gene in the growth and development of G. daurica. [Methods] Based on transcriptomic data, the full-length cDNA sequence of the G. daurica TPP gene was cloned using the RACE method, and analyzed with bioinformatic software. The gene was then inserted into the prokaryotic expression vector pET-28a (+) to create a recombinant plasmid pET-GdTPP, which was then transformed into Escherichia coli BL21 (DE3) to express TPP. qPCR was performed to profile the expression of the gene in 2nd-instar larvae under different temperatures. [Results] The full-length cDNA sequence of the G. daurica TPP gene (GdTPP, GenBanka accession No: MG431210) was obtained from G. daurica larvae. GdTPP is 1 372 bp in length with an open reading frame (ORF) of 864 bp, encoding 287 amino acids and has a predicted molecular weight of 32.32 ku and pI of 6.19. The encoded protein contains two potential N-glycosylation sites. Subcellular localization prediction indicates that the encoded protein is located in the cytoplasm and lacks signal peptide and transmembrane domains. GdTPP has one conserved domain, and is most similar to Leptinotarsa decemlineata TPP in amino acid sequence identity (68%). A recombinant plasmid, pET-GdTPP, was successfully constructed, and GdTPP was efficiently expressed in E. coli. The qPCR results show that, at temperatures lower than 25 ℃ (control), the expression level of GdTPP increases with decreasing temperature to a maximum of 1.86 times that of the control at ﹣10 ℃. However, at temperatures higher than 25 ℃, the expression increased with temperature to a maximum of 1.68 times that of the control at 40 ℃. [Conclusion] The expression of GdTPP in G. daurica larvae was up-regulated in response to cold- and heat-stress. These results provide a basis for further investigation on the role of TPP in temperature stress in insects.