The Construction Of Indigo Gene Expression Vectors And The Study Of Cloning Carnation CHS Promoter

With the development of mordern lives, more and more flowers are needed and that indicates a prosperous prospect of ornament market. There are some restricting factors in traditionary breeding methods, and nowadays the molecular breeding methods are well developed and have succeeded in some flowers such as chrysanthemum and lily et al.In this paper, we introduce our study on the cloning of two petal-specific CHS (chacone synthase) promoters and the constructing of expression vectors containing of a CHS promoter and an indigo gene (bec gene) or a GUS report gene. The main contents are as follows:(1) We have cloned two CHS promoters from Arabidopsis thialiana (Columbia ecotype) using two pairs of sequence-specific PCR primers from two homologous sequences in the Genbank (AF248988 & AF012810). The fragments are 533bp and 2046bp separately, and the cloning sequencing results indicate that the homologous between them and the Genbank sequences excess 99%. We have also analysised the two promoters' construction using PlantCARE software on the internet, the results reveal that they have the motifs that are necessary for eukaryotes promoters such as TATA box and CAAT box et al. and they also have some motifs that are specific in CHS promoters. The results prove that these two promoters should have promoter activity.(2) By analysising the restriction endonucleases sites of the two promoter sequences and the vectors, we selected the profitable restriction endonucleases to construct the expression vectors. The two promoter fragments are ligated to a GUS report gene or a bec gene separately. The two expression vectors containing the shorter promoter have already been finished and the other two vectors containing the longer promoter are on finishing.(3) The aim of our experiment is to get high efficient expression vetors to express the bec gene in ornaments. For fearing that the CHS promoters from Arabidopsis maybe couldn't promote the gene expression in ornaments, we tried to clone a CHS promoter from carnation. But the homologous between the CHS promoters of different plants are very low, so we designed a pare of PCR primers from carnation CHS gene sequence and have got three clones of carnation CHS gene 5' end sequences by PCR. With these three sequences we used adapter ligation PCR method to clone the promoter, and now we have got two fragments which are about 500bp and about 800bp separately, the cloning sequencing of them are on finishing. The results prove that adapter ligation PCR method is workable to clone carnation CHS promoter. And the next step we will do the deletion analysis of the promoter fragments.(4) We have also done some experiments on establishing the regeneration system of carnation, and have selected several kinds of comparativly ideal cultures. The following works are on proceeding.