Truncation Modification Of Trehalose-6-phosphate Synthase Gene (SpTPS1)in Selaginella Pulvinata

Trehalose synthesis is catalyzed by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase(TPP) and trehalose-6-phosphate synthase is the rate-limiting enzyme. Transformation by the fusion genes of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) from E. coli and brewer’s yeast into tobacco,potato,rice and Arabidopsisto gain the transgenic plants showed that the abiotic resistance (such as drought tolerance)of them had improved greatly.However,over expression of the TPS genes caming from E.coli and yeast led to deformity phenotype in transgenic plants,while the endogenous TPS gene in plant did not appeared these phenotypes.These phenomenons revealed that the TPS gene caming from plants had broader application prospects in transgenic plant research compared with those from E. coli and yeast.In pteridophytes, especially some xerophytic species in Selaginellaceae, the trehalose content can be accumulated as high as20%under drought conditions.American scientists have cloned the SlTPS1gene from Selaginel lalepidophylla, and had applied for patent protection. However, TPS activity from plant is lower and also there is little trehalose accumulation in yeast expression.Compared with TPS enzyme identified in E. coli and yeast, the amino acid sequences of TPS enzyme encoded by TPS gene in Selaginella lepidophylla contains an extra plant-specific extension at the N-terminus.The truncation modification of the N-terminal extension resulted in higher TPS activity. Our research group successfully cloned the TPS gene from Selaginellapulvinata, named SpTPS1,through the homologous amplification method. The TPS1deletion mutant strain of the yeast has been transformed by the SpTPS1gene and we have applied for patent protection.In this study,we did multiple alignment between the putative amino acid sequences of SpTPS1gene and the eight species (Selaginella lepidophylla, maize, Arabidopsis thaliana, Oryza sativa, Saccharomyces cerevisiae, Triticum aestivum and E.coli) which containing TPS1cDNA sequences. It showed that the N-terminal has75 amino acids for its extended portion, so we designed the corresponding primer and used PCR amplification for the truncated modification of trehalose-6-phosphate synthase gene. Then we constructed the yeast expression vector and transformed yeast TPS1mutant to do functional identification of the TPS gene. By comparing with catalytic activity of TPS between full sequences gene and the truncated modificated gene, the results were as followed:(l)Successfully cloned2970bp length gene (SpTPSl) and the truncated225bp modified gene (SpTPSlΔ);(2)Yeast heterologous complementation experiment proved that the two genes encoding proteins have trehalose-6-phosphate synthase function;(3)The TPS activity of the mutant strain transformed by the truncated gene SpTPSl Δ was about six fold higher than that transformed by its original version,and after heat shock the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only8%higher than that transformed by its original version.After transform the truncation modificated trehalose-6-phosphate synthase gene SpTPS1Δ to corn and other crops, the abiotic resistance (such as drought tolerance) may be improved more obviously.