Detection Of The Trehalose And Cloning And Sequence Analysis Of Trehalose-phosphate Synthase Gene In Maize

Maize is the second crops in China.Maize is a special variety that needs a lot of water and is sensitive to the water stress.In China,about 60%of maize planting area are harmed by drought,and annual losses are appraised at 15%-20%,just inferior to the losses caused by plant diseases and insect pests.So,studying the mechanism of maize in tolerant to drought and developing new variety to resist drought,is a effective way to reducing the harm of drought to maize production.A lot of trehalose was produced in Selaginella lepidophylla on the drought condition.The stress-protective properties of trehalose in vitro on proteins,membranes and intact cells have been amply documented.Trehalose was discoveried in other plants such as Arabidopsis thaliana,Oryza sativa et al,and had some correlation to drought.Some scientises have used the TPS/TPP gene transgenie into Arabidopsis thaliana,Oryza sativa and tobacco to improve the drought-resistant ability of plants.The trehalose amount was quantified by high performance liquid chromatography(HPLC).the contents of trehalose was10.31mg/g dry weight in Selaginella lepidophylla.But there was no visible trehalose in maize and wheat.In this study,I used the tps cDNA of Oryza sativa as the querying probe,and highly homologous EST sequences were blasted in dbEST database of maize at Genbank. The downloaded EST sequences were aligned and assembled into contig by Seqman program of DNAstar software.To validate the correction of the splicing sequence,the specific primers were designed and used to amplify the complementary sequences of the assembled contig by RT-PCR,According to the sequencing contig,open reading frames(ORF) were analysed by ORF Finder program at NCBI website.The full length cDNA sequence of ZmTPS gene was obtained by using rapid amplificationof cDNA ends(RACE) technology.The results showed that the cDNA of ZmTPS gene was 3055 bp,encoding897 amino acid residues,and deduced nucleic acid an amino acid sequence possessed high identity with higher plants.Alignment showed that the putative amino acid sequences had 52.26%and 41.56%similarity with tps genes in Arabidopsis thaliana and Oryza sativa,and had a conservative region of TPS.ZmTPS gene might have the function of TPS.We found that the sequences of ZmTPS had a some amino acid at N termination as similar as Arabidopsis thaliana and Selaginella lepidophylla,these amino acid might inhibit the activity of TPS(Van Dijck et al,2002).Some plants had a little of trehalose,because of these TPS sequence lost some important amino acid,such as GGL motif and some amino acid which link to substrate.These factors that degraded the activity of TPS caused little trehalose in plant.