Construction Of Inducible Ac/Ds Transposon Systems And Analysis Of Their Transposition Behaviors In Arabidopsis Thaliana

At present, transposon tagging is one of the main ways for mutant screening and gene cloning. In transposon tagging methods, Ac/Ds (Activator/Dissociation) tagging system is an ideal method to build mutant library. Ac/Ds system is the member of hAT family, including the independent transposable element AcTPase and the dependent transposable element Ds. AcTPase element can transpose under the activity of transposase encoded by itself. However, it transposes constantly and it is difficult to gain stably transposed plants, making it not very convenient to study or use in research. Therefore, in our study, the Ac element is changed and lost the ability of transposition, keeping the ability of encoding transposase. Ds element does not posess the ability of encoding transposase, so it only transposes in the presence of Ac ele ment. The inducible expression of transposase makes the transposition process under control, increased the efficiency significantly, and making the insertion events stable. The main results are as follows:1) Constructed the ethanol inducible Ac/Ds transposon system pHL1 and dexamethasone (Dex) inducible Ac/Ds transposon system pHL2. Chemical inducers of ethanol and Dex make AcTPase gene express transposase, making Ds element transpose effectively in specific devel opment stage in transgenic plants.Ethanol inducible system includes alcR that encodes transcription factors ALCR and eth anol inducible promoter alcA. alcA promoter can't promote AcTPase gene expression without ethanol. When ethanol are added, they bind to ALCR proteins and change their conformation, making them bind to alcA promoter, and starting to express AcTPase.In Dex inducible system, cDNA of AcTpase gene was fused with GR-glucocorticoid rec eptor gene, expressing fusion protein. This protein locates outside the nucleus, combining to heat shock proteins without activity. When steroid ligand is added, fusion protein releases from complex, then comes to the nucleus, and acts on the recognition sites, causing the Ds element transposition. The steroid ligand is dexamethasone in my experiment.These two vectors both contain GFP, GUS reporter genes. GFP gene locates in the Ds element, so GFP fluorescence reveals the existence of Ds-GFP element, then it is easy to detect the excision and reinsertion of Ds-GFP. pCaMV35S promoter and GUS reporter gene are located at the ends of the Ds element. The excision of Ds causes the expression of GUS gene, so the transposition of Ds can be detected by GUS staining. 2) pHL1 vector was transformed into Arabidopsis thaliana, and single copy lines were recovered. Ethanol treatment experiments show that the expression level of transposase in induced plants are stronger than that in noninduced plants through semi-quantitative poly merase chain reaction (PCR). The results of GUS staining, nested PCR from the donor site of Ds, show that Ds-GFP element transposed. In the progeny of ethanol challenged plants stable transposition events have been produced.3) The Dex inducible Ac/Ds system was introduced into Arabidopsis thaliana and single copy homozygotes were produced. GUS staining analysis suggested that Ds-GFP could trans pose in Arabidopsis and stable transposition event has been obtained in progeny of plants treated with Dex.