Cloning And Functional Analysis Of Trehalose-6-phosphate Synthase Gene (SpTPS1) From Selaginella Pulvinata

Water is the fundamental material in plants, drought have a wide range of impact on plants. In drought stress conditions, plant metabolism has changed, physical activities blocked, the various stages of growth impacted, and appears wilt phenomenon.Selaginella pulvinata, commonly known as the "resurrection grass", displays extremely tolerance to desiccation. It can survive in the dehydration and re-hydration cycle, the curling yellow parts can be quickly extended and resurvived once got water. Studies have shown that the "resurrection plant", such as Selaginella pulvinata, contains a lot of stress metabolites-trehalose. Trehalose plays a special protective role in organisms, it can maintain the biological activities, even in the abnormal conditions, such as high temperature, dehydration and freeze. The trehalose synthesis pathway, which uses UDP-glucose and 6-phosphate glucose as substrate, is the most widely distributed in eubacteria, fungi and plants, the Trehalose-6-phosphate synthase, encoded by TPS1 gene, is the key enzyme in the pathway. The researchers have been cloned TPS1 in plants, but they found there is no accumulation of trehalose in vivo, except for the resurrection plants. They also have been transformed the S. cerevisiae TPS1 and E.coli otsA into plants, the positive plants increased the trehalose content in vivo and tolerance to desiccation, but had varying degrees of changes in development and morphology.It is a transgenic techno-key to gain the higher drought-resistant gene that is fit for corn physiological mechanisms and production. Our research chose the extremely drought-tolerant Selaginella pulvinata to clone trehalose-6-phosphate synthase gene (SpTPS1) by RACE, the gene whole length 3223 bp, containing 2790 bp open reading frame, and analyzed the nucleotide and mature protein through bioinformatics.To research the SpTPS1 gene function, we subcloned the ORF into vector pUC119, and then select the appropriate restriction site to respectively digest the vector OpUC119 and pRS6/AtTPS1, the ORF fragment and the pRS6 framework from pRS6/AtTPS1 were linked, and finally we got the eukaryotic expression vector OpRS6. Transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 into the host yeast strain tps1â–³, YSH290 by lithium acetate, and then spread the dilution onto a selection medium which contains the appropriate galactose and lacks Histidine. The result shows that wild-type strain W303-1A and deletion yeast strain tps1â–³can not grow in the medium; but the yeast strain which transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 can grow in the medium. We substituted 2% galactose for 2% glucose in the selection medium, and selected the positive transformants to grow on the medium. The result shows that the wild-type strain W303-1A and deletion yeast strain tps1â–³can not grow in the medium which contains glucose and lacks Histidine; but the yeast strain which transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 recoveried the function of deletion yeast strain tps1â–³, and can grow in the medium containing glucose. Extracted the plasmid DNA of the positive strains which is testified correct by PCR detection. Consequen -tly the SpTPS1 gene can be testified to encode the active trehalose-6-phosphate synthase by the yeast fuctional complementary test.In this study, we cloned drought-tolerant gene SpTPS1, it is valuable for a wide range of applications to cultivate new drought-resistant varieties. It provide a higher drought-tolerant gene that is fit for corn physiological mechanisms and production to cultivate new drought-tolerant transgenic cores.