Preliminary Functional Analysis Of Dicarboxylate-tricarboxylate Carrier Protein Gene (CjDTC) From Citrus Junos

Development of acid soils that limit crop production is an increasing problem worldwide. Many factors contribute to phytotoxicity of acid soils, however, in acid soils with a high mineral content, aluminum (Al) is the major cause of toxicity. Al can limit the grows of many plants at micromolar concentrations. Organic acid be capable of chelating and forming a stable complex with Al that harmless to plant, therefore, Al-induced efflux of organic acid from plant roots has been considered as an important mechanism for Al tolerance. As the intermediates of TCA cycle, organic acid is abundance in mitochondrial. To transport organic acid from mitochondrial to cytoplasm, a carboxylate carrier family is needed. Previous researches revealed that tight link between the process of organic acid transport from mitochondrial and release organic acid to rhizosphere.To reveal the role of carboxylate carrier protein in the mechanism of Al tolerance in plants, the CjDTC, which encoding dicarboxylate-tricarboxylate carrier protein gene of Citrus junos, had been obtained. In this thesis, the upstream promoter sequence of CjDTC have been cloned. The promoter of CjDTC drive GUS was transformed into tobacco mediated by Agrobacterium, and the histochemical analysis of CjDTC∷GUS transgenic tobacco lines were carried out. Beside that, We also analysised the expression level and Al tolerance of overexpression of CjDTC in tobacco.The main results were as follows:1. The upstream promoter sequence was obtained by YADE technique, and it was fused with GUS gene to construct expression vector. The vector was transformed into tobacco mediated by Agrobacterium. The CjDTC promoter driving GUS transgenic tobacco lines were obtained by PCR, and its GUS activity were induced in the root with Al³⁺(pH4.3).2. Northern blotting analysis indicated that the CjDTC was induced in the roots with Al³⁺ (pH4.3) solution.3. The CaMV 35S driving CjDTC constitutive expressing vector was introduced into tobacco, and three transgenic plants of over-expression were obtained through Northern-blot.4. Three T₂ homozygous lines were used to assay its tolerance to Al³⁺. Eriochrome cyanine R staining analysis showed that the ability of decrease Al accumulation in transgenic tobacco is stronger than wildtype at early stage of Al toxicity. However, the relative root growth and content of H₂O₂ was insignificant difference between transgenic tobacco plants and wildtype with 100μmol/L Al³⁺ (pH4.3).Taken together, CjDTC is involved in the mechanism of Citrus junos Al tolerance. Overexpression of CjDTC may improve the plants Al tolerance, but enough and detailed data are still further supplied.