Improvment On The Plant Promoter-analysis Vectors In Recombination Cloning

Promoter is a kind of DNA region or sequence that can combine with RNA polymerase and other transcription factors. The promoter- analysis is a very important and efficient method used in genes function-analysis and specific expression research. First we constructed 17 promoter-analysis vectors of 12 different gene promoters using the traditional cloning system based on restriction enzymes and ligase. Though most of the constructions seem to get successful results, we still found lots of existent and potential problems through deep analysis of these sequences and transgenic material's Gus staining results. The 5' untranslation region, segment length, especially the TATA box and the putative ATG between the native ATG and the transcription start site have important influences on the final success. The 2×35S promoter of the resistant gene upstream also interfere our experiment reliability. Besides we also found that the transgenic lines were so distinctive to each other that it is hard for us to get an unique and reliable result.Now we have transferred to a new cloning system called Gateway Technology^tm in order to get better efficient promoter-analysis vectors. Whereas the fact known so far that the transgen variation mainly raised by the distinct chromatin environments and various activate or inhibit signals especially the heterochromatin surrounding the different insertion sites. While insulater is a kind of DNA barrier element that possesses a common ability to protect genes from inappropriate signals emanating from their surrounding environment. So it is widely used in transgens in order to minimize the transgenic variation. In fact, it has been used successfully and widely in transgenic mouse, rabbit and cell lines. However because of the later beginning in plant insulator research, it has few reports about the applications of insulators in plant transgens. In this paper, we did such a pathbreaking work by adding a drosophila insulator sequence to our Gateway promoter-analysis vector. The results are very heartening that this insulator significantly decrease the transgen variation in transgenic Arabidopsis as we expected and even provoke the Gus expression. We also construct another new vector by adding a translation leader sequence to the 5' of Gus gene. The results also meet our expection. And at last we construct three other new vectors by inserting report genes flanking the insulator sequence, hoping that the insertion segment can decrease such interference, and meanwhile provide us some information about this insulator function in Arabidopsis through the fluorescence results.