Structure, Function And Regulation Analysis Of Cotton Sucrose Synthase 3 Promoter

Cotton fibers play an important economic role in the world, it supplys the crude fiber for textile. Each cotton fiber is a highly elongated single cell of maternal origin that initiates from the ovule epidermis outer layer at or just before anthesis. It is divided into four distinct but overlapping phases: initiation, elongation, secondary wall synthesis and maturation. About 15-25%of the epidermal layer cells differentiates and develops lint fibers. In recent decade many studies were focused on molecular mechanism of the cotton fiber development. The cotton sucrose synthase 3 (SuSy) is one of the functional genes which were found involving in cotton fiber cell initiation and elongation, plays a role in carbohydrate partitioning and ovule development. It catalyzes a reversible reaction from sucrose to fructose and UDP-glucose, and the latter is the prefect substrate for cellulose biosynthesis in cotton fiber. Several studies have indicated that SuSy performed a very important role during the fiber initiation and elongation. Furthermore, down-regulation of SuSy mRNA levels in the cotton ovular epidermis by RNAi leads a fiber-less phenotype.Efforts to understand and manipulate fiber development would be enhanced by an improved knowledge of the promoter elements and transcription factors that regulate fiber relative gene expression. The structure and genetic control of trichome morphogenesis of Arabidopsis have been extensively characterized, showing a number of structural and genetic similarities to cotton fibers. Thus, it has been possible to monitor the activity of cotton fiber relative promoters in this hetreologous model specie. The results are described as following:1. According to the sequence of SuSy cDNA (U73588), genomic DNA of this gene was isolated by PCR. There are 13 exons in the gene coding region and its genomic structure has a high similarity with Sus1 and Sus4 of Arabidopsis thaliana in the length of exons;2. A1554bp promoter fragment was isolated by LA PCR^TM in vitro cloning kit after three times PCR- based genome walking. The promoter then fused to GUS gene with a part of the first exon of SuSy and three binary vectors related to intron were constructed. All resulting vectors were transformed into Arabidopsis thaliana mediated by agrobacterium;3. The transgenic plants demonstrated highly specific GUS expression in the vascular tissue, trichome, cotyledon, root, root hair and flower. The region between -554 and -1554 enhanced the GUS activity to more than 20 fold more than that of 554bp promoter fragment;4. GA₃ could make the GUS activity of 1152bp promoter fragment (Susp8::1301) increasing 2.5 times, but has no effect on that of the whole promoter (Susp9::1301)and the other fragments less than 810bp (Susp7::1301, Susp6::1301, Susp5::1301, Susp4::1301);5. The fragment from the -296 to -396 is essential for the promoter activity in transgenic Arabidopsis trichome, and also play a role in driving GUS expression in pollen;6. Mutation of a W-box in the position of -96bp reduced the GUS activity in transgenic plants, but no changes were found of the promoter tissue specificity;7. The intron specific inhibited the GUS expression in mature pollen and reduced 90% GUS activity in other tissues;8. GA₃ enhanced the GUS activity in Susp9fi::1301 transgenic plant with 4 fold.