Isolation And Characterization Of GaP5CS And GaTPS In Gossypium Arboreum L.

Osmotic adjustment is one of the most important ways that higher plant devolepes to maintain the osmotic balance and resist to environmental stress under water deficiency.Proline and trehalose are two improtant osmolytes.Using the method based on homologous sequences and bioinformatics,the P5CS gene,coding for pyrroline 5-carboxylate synthetase,and the TPS gene,coding for trehalose 6-phosphate-synthase,were isolated and characterized from Gossypium arboreum(Shixiyaâ… cultivar,known for its drought tolerance).Both of the two enzymes are the key enzymes in the biosynthesis of osmolytes-proline and trehalose repectively.The following results were achieved through this study:1 Gene cloning and sequence analysisUsing RT-PCR method to obtain the coding region of genes, combined with 3 'and 5'RACE technology,3'-UTR and the 5'-UTR were obtained.In the end ,we use the sequence splicing to obtain full-length cDNA. Gossypium arboreum P5CS(2827bp) consisted of an open reading frame of 2148bp,a 5'untranslated region of 350bp ,stop codon TGA and 3'untranslated region of 326bp.GaP5CS is a biofuncational enzyme which consisted of two domains: glutamyl kinase domain and glu-5-semialdehyde dehydrogensae domain. Gossypium arboreum TPS(3219bp) consisted of an ORF of 2583bp,a 5'untranslated region of 255bp,stp codon TGA and a 3'untranslated region of 378bp.GaTPS is also a biofuncation enzyme which consisted of two domain:trhalose-6-phosphate-synthase and trehalose-6-phosphate-phosphatase.2 Gene expression analysisWe use 17%PEG6000 to deal with Gossypium arboreum seedling 3-6 leaves to simulate drought stress conditions.RT-PCR analysis showed that the transcriptions of the GaP5CS and GaTPS genes were strongly induced by 17%PEG6000.Thus,we speculate that GaP5CS and GaTPS are likely to play an important role in the process that Gossypium arboreum respond to drought stress.3 Analysis of genes functionGaP5CS and GaTPS were transformed into Nicotiana babacum by Agrobacterium infiltration method respectively in order to test their functions.Transgenic lines had lower water loss rate and lower percentage of ion leakage under drought stress,which showed that GaP5CS and GaTPS inhanced the drought-tolerance of Nicotiana babacum.Then we transformed the two genes into Gossypium hirsutum L.,using Agrobacterium-mediated transformation and particle bombardment transformation,so as to the further in-depth study was going on to verify these two genes may enhance the cotton drought-tolerance.This research was the pilot work about the GaP5CS and GaTPS genes in cotton(Gossypium arboreum L.),thus providing alternative genes for molecular genetic improvement of cotton drought-tolerance and providing the theoretical basis for cultivating new varieties of drought-tolerant cotton.