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Construction Of Inducible Guard Cell-specific Promoters And Effects Of Overexpression Of Atdof1.7 On Stomatal Movements

Posted on:2005-10-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:J LiFull Text:PDF
GTID:1100360122488895Subject:Botany
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Stomatal pores on the surface of leaf are gateways of gas exchange between plants and atmosphere. It was estimated that plants account for around 65% fresh water use every year, which was mainly lost through stomata. So it is necessary to regulate the opening and closing of stomata in order to save water and improve plant drought resistance. The genetic regulation of stomatal movement mainly depends on an efficient control system of gene expression, and guard cell-specific promoter is becoming one of the best choices. Then a tentative plan came to our brains, is it possible for us to assure guard cells to open when water supply is enough, and close rapidly when plants come across drought stress by regulating expressional pattern of genes? Inducible guard cell-specific promoters can play an important role in driving target gene expression at specific position in given conditions such as drought stress. In this study we constructed two promoters (DGP1 and SIGP1) and analyzed their function in transgenic tobaccos.We combined the dehydration responsive element (DRE) with guard cell specific element (GCSE) to construct a novel drought-induced guard cell-specific promoter -DGP1. Histochemical assays in transgenic tobacco carrying B -glucuronidase (gus) gene fused to DGP1 showed that GUS activity was found to be highly inducible by drought treatment and specifically restricted to guard cells. No GUS activity was detected in roots, stems or flowers after treatment. Further quantitative analysis showed that GUS activity in the epidermal strips was apparently induced by dehydration and increased dramatically with the prolongation of treatment time. The GUS activity after 8 hours' treatment was 179 folds compared with that of those without treatment and no great changes were observed in roots, stems or mesophyll. These results suggested that DGP1 could drive target gene expressed mainly in guard cells when plant is subjected to drought stress.We constructed a multi stress induced promoter (SIGP1) strongly expressed in guard cells. The full length of S1GP1 is 746 bp, containing a number of TCGT motifs, three 5'-TAAAG-3'core sequence and a TATA box. Histochemical staining in Tl transgenic tobaccos showed that SIGP1 promoter was not only organ-specific (especially expressed in leaves and guard cells), but also highly inducible by multi stress treatment such as drought, ABA, high salinity and low temperature. Quantitative analysis showed that GUS activity in roots was very weak and no great changes were observed after treatment. However, GUS activity in leaves and epidermal strips increased with the prolongation of treatment time.In order to further understand the regulatory mechanism of stomatal movement, we studied one of transcription factors that might be related to stomatal movement. Using RT-PCR we isolated Atdofl.7 from Arabidopsis, which had a higher homology with StDOFl whose expression was very high in guard cells. However there was little knowledge about the function of Atdofl.7, especially to stomatal movement. We constructed plant expression vector driven by CaMV35S promoter and obtained Tl transgenic tobaccos. The results represented upon observation of stomatal movement showed that the density of stoma in the epidermal of transgenic tobaccos was much lower than that of wild type and therate of opening and closing of stoma increased because of strong expression oi Atdofl. 7. It can provide the theoretic basis to study the stomatal movement by regulating the expression of transcription factors.
Keywords/Search Tags:guard cell-specific promoter, inducible promoter, DOF transcription factors, Atdof1.7, tobacco, Arabidopsis thaliana
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