Genetic Transformation And Functional Characterization Of Pitaya CAT Gene In Tobacco

Abiotic stresses severely affect distribution, growth, development and quality formation of plants. Subjected to abiotic stresses, ROS of plants is substantially accumulated, thereby leading to severe damage to cellular components, e.g.membrane and DNA. Catalase(CAT), belonging to hemoglobin enzymes mainly catalyze the conversion of H202 to H20 and 02 in plants. Over-expression of CAT can improve oxidative stress tolerance by efficiently scavenging ROS produced in plants.Pitaya(Hylocereus undulatus), which originated from tropical desert terrain of Central America, is fruit tree of cactaceous plants, and is of great economic value.Previously, a CAT gene(Hu CAT) was cloned from pitaya and preliminarily justified involvement in abiotic stress tolerance. In current work, plant expression vector of Hu CAT were constructed and transformed into tobacco. To further investigate the potential role of Hu CAT in plant abiotic tolerance, physiological indexes of transgenic plants subjected to drought and low-temperature stress were determined. The main results were as follows:1. Based on our previous work, Hu CAT was amplified from cloning vector PMD18T-Hu CAT using specific primer pair. Products were then inserted into vector p SH737 to construct sense and anti-sense plant expression vectors promoted by Ca MV35 S.2. Using Agrobacterium tumefaciens-mediated co-transformation method, 37 clones were harvested from the leaf discs of tobacco(‘Xanthin'), among which, 29 were proven to be transgenic lines after GUS gene histochemical staining and PCR detection. With semi-quantitative PCR and real-time quantitative PCR detection, 22 transgenic lines over-expressing Hu CAT were obtained.3. Several physiological and biochemical parameters involved in oxidative stresswere investigated in the transgenic tobacco subjected to PEG-6000 treatment. The results showed that the enzyme activities of CAT, SOD or POD, the contents of chlorophyll, soluble sugar, soluble protein and PRO, as well as the relative content of water were higher than that of the wild type. On the contrary, the contents of MDA,H2O2 and relative electrical conductivity in the transgenes were lower than that of the wild type. Therefore, the transgenic tobacco plants over-expressing Hu CAT could substantially quench the oxidative status of the drought-stricken plants.4. The physiological and biochemical parameters implicated in oxidative stress were also determined between the transgenic plants and the wild type as exposed to low temperature(4 ?) for 2 days. The results demonstrated that the enzyme activities of CAT, SOD or POD, as well as the contents of soluble carbonhydrate, soluble protein or PRO in the transgenic plants were higher than those in the wild type.Conversely, the MDA accumulation in the transgene was lower in comparision with the wild type. Therefore, Hu CAT could improve the cold resistance of tobacco.Conclusively, over-expression of Hu CAT may considerably ameliorate the oxidative stress via ROS scavenging, leading to enhancement of adversity stresses such as water deficit and low temperature.