Construction Of Inducible Ds Transposition Vectors And Their Application In Arabidopsis And Maize

The selection and identify of mutant is one of the main way to develop molecularbiology，and currently the transposon tagging screening mutant and cloning genes is one offunctional genes mainstream method. Ac/Ds (Activator/Dissociation) tagging system is anideal method to build mutant library. Ac/Ds system is the member of hAT family, includingthe 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 veryinconvenient to study or use in research. Therefore, in our study, the Ac element is changed，we made it lost the ability of transposition and keep the ability of encoding transposase.The Dselement does not posess the ability of encoding transposase, so it only transposes in thepresence of the Ac element. The inducible expression of transposase makes the transpositionprocess has been controlled, can significantly improve the frequency of transposition process,and can quickly stable insert events.We all know that the Arabidopsis thaliana still have30percent genes that their functionsunkouwn.In our research,we supply a convenient way to study the unknown gene byenriching the event that Ds transpose and reinsert.We first identify the function of twovectors，then applied in maize。Also，it will be useful in improving the quality of crops.The main results are as follows:1) Identify the inducible function of the two vectors. pHL1vector was transformed intoArabidopsis thaliana, and single copy lines were recovered. Ethanol treatment experimentsshow that the expression level of transposase in induced plants are stronger than that innoninduced plants through realtime quantitative PCR. The Dex inducible Ac/Ds system wasintroduced into Arabidopsis thaliana and single copy homozygotes were produced. GUSstaining analysis suggested that inducible function is good.2) Identify the transposable function of the two vectors. pHL1vector was transformed into Arabidopsis thaliana, and single copy lines were recovered. After the ethanolinducble,the results of GUS staining, nested PCR from the donor site of Ds, show thatDs-GFP element transposed.We identify the insert sites by Tail PCR,inverse PCR and adapterPCR．The Dex inducible Ac/Ds system was introduced into Arabidopsis thaliana and singlecopy homozygotes were produced. After the Dex inducble，GUS staining analysis suggestedthat Ds-GFP could trans pose in Arabidopsis．We identify the insert sites by the adapter PCR．3) The stable transposition event has been obtained in progeny of plants treated withEthanol and Dex. After inducble we obtained the stable transposition event by GUS staininganalysis.4) We verified the feasibility and the application of the two inducible vectors，provideda convenient and feasible method for better researching other functional genes in plants.