Isolation And Characterization Of GaP5CS Promoter And GaTPS Promoter From Gossypium Arboreum L.

Constitutive promoters, such as CaMV 35S, were mostly used in transgenic plant engineering. They drive foreign gene to express during the whole growth period and in every organizations of transgenic plant, which often causes substances and energy waste, increases the metabolism burden of plant, even leads to changes of plant morphology and affects plant growth and development. Therefore, using tissue specific promoter or inducible promoter can not only improve level of expression of the foreign gene, increase the effect of genetically modified, but also reduce plant energy consumption and the damages of plant.Proline and trehalose are two important osmolytes. Pyrroline-5-carboxyate synthetase and trehalose 6-phosphate-synthase are the key enzymes in the biosynthesis of proline and trehalose, respectively. The promoters of the two genes were cloned and characterized from Gossypium arboreum(Shixiyaâ… cultivar).The main results were as follows:1 Promoter cloning and sequence analysisUsing modified TAIL-PCR method, the two genes 5'upstream sequence were amplified from the Asian cotton genome, which named GaPROP5CS and GaPROTPS. Sequence analysis showed that GaPROP5CS was 795bp in length, in which the TATA box was found at position -22 relative to the putative transcriptional star site (TSS). GaPROTPS was 1269bp in length and TATA box was at 32bp away from the TSS.2 Transient expression analysis of promoterTwo expression vectors were constructed, in which GUS was drove by GaPROP5CS and GaPROTPS, respectively. The vectors were transformed into onion by particle bombardment transformation to test their functions, respectively. The onion were cultivated in the medium with PEG10%.The results suggested that GaPROP5CS and GaPROTPS are the drought stress-induced promoters, and the activity of GaPROP5CS is stronger than that of GaPROTPS.3 Analysis of promoter functionGaPROP5CS and GaPROTPS were transformed into Nicotiana babacum by Agrobacterium infiltration method to test their functions, respectively. Under drought stress, gene expressions of GUS were found in leaves and roots of transgenic plants, GUS activity showed that the two promoters had a stronger expression of activity after drought stress-induced. To further study the expression activity of GaPROP5CS, we transformed the GaPROP5CS into Gossypium hirsutum L., using Agrobacterium- mediated transformation.4.Deletion analysis of promoterTo study the negative regulatory element, a series of 5'-deletions were legated to GUS in expression vector. Then, we transformed the deletions into onion by particle bombardment transformation. Lastly, the transgenic onions were cultivated by PEG. The GUS activity results showed that the highest GUS activity were examined at the region of 444/+52 and -123 /+52, which were equal to CaMV-35S activity. Then, we transformed it into Nicotiana babacum by Agrobacterium infiltration method, so as to select the deletion of highest activity.