Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata.
A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225bp at the 5'-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.
Maize Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, People's Republic of China.
Sheng-Mei
SM
Zhao
Feng-Ling
FL
Fu
Lin
L
Gou
Han-Guang
HG
Wang
Gang
G
He
Wan-Chen
WC
Li
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005816:Genetic Complementation Test; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D016366:Open Reading Frames / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D032503:Selaginellaceae / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D014199:Trehalose / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D014199:Trehalose; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase