The Study On The Mechanism Of CAT Overexpression Effected Formaldehyde-uptake Ability Of Tobacco

Modern industry make a lot of HCHO used in decorating material, which make HCHO as the primary pollutants indoor pollution in our country. Formaldehyde governance has become the main problem of indoor air pollution. In numerous HCHO treatment methods, the plant purification method for its advantages of environmental protection, economic, convenient that is noticed and favored by people. Common ornamental plants because of their different formaldehyde absorption capacity and are generally lower, so the effect of the removal of HCHO is not very ideal. There are studies have shown that plant produce a lot of H2O2 when stress by gas HCHO, and the H2O2 induced a series of signal transduction pathways to promote stomatal closure, and reduces the absorption efficiency of the plant to HCHO. This research attempts to import CAT (Catalase) gene of arabidopsis in wild type tobacco chloroplast, which can increase the activity of antioxidant enzyme of the transgenic tobacco. When plants are suffered from HCHO stress, which can timely and effective to remove the accumulation of H2O2 in plants and keeping stomata larger stomatal opening and conductance, to promote the plants absorb efficiency of HCHO. For the use of genetic engineering to improve plants HCHO repair ability to provide new operational policy and theoretical basis. The main results were as follows:1. In order to make the CAT expression then positioning in the chloroplast, Using the Gateway technology we structure a plant expression vector pK2-PrbcS-*T-CAT that CAT expression driven by photoinduced type promoter (Prbcs). To express the CAT protein through the vector is guided to the chloroplast of plant by stroma positioning signal peptide (*T) downstream Prbcs.Using pK2-PrbcS-*T-CAT transformed to wild type (WT) tobacco and then obtain five gm strains of kanamycin (Kan) resistant. Genome PCR analysis confirmed that five resistant strains all have CAT gene inserted. RT-PCR analysis confirmed that five transgenic strains all have CAT gene expression, then determination of CAT activity selected of transgenic tobacco strains C1 and C3 of different CAT activity.2. Analysis of WT and C1, C3 transgenic tobacco treated with high concentration gas formaldehyde then the influence of the physiological characteristics. Results show that 10 and 20 ppm of gas HCHO stress after a short time, the stomatal conductance and opening of C1 and C3 transgenic tobacco are bigger than WT, and the oxidative damage indexs include H2O2, MDA and PC were less than WT. H2O2 fluorescent probe analysis confirmed that the number of the the guard cells of blade under skin in Cl and C3 which accumulation of H2O2 was significantly less than WT under 10 ppm gas HCHO stress. The gas HCHO absorption efficiency of C1 and C3 transgenic tobacco under 10 and 20 ppm is higher than that of WT. Guard cells have chloroplasts and chloroplast is a source of the H2O2 in the guard cells, and then excessive expression CAT may by lowering C1, C3, the accumulation of H2O2 in the guard cells of Cland C3 that increased leaf stomatal conductance and the opening under gas HCHO stress, thus increasing their gas HCHO absorption efficiency. Immune coprecipitation and Western analysis that under 10 and 20 ppm gas HCHO stress, the 14-3-3 protein and plasma membrane H+- ATPase of transgenic tobacco was higher than WT, and the activity of plasma membrane H+-ATPase and hydrogen pump were all higher than the WT. It was demonstated that transgenic tobacco can remove H2O2 which was excess accumulation, and which can terminate the signal transduction of stomatal closure caused by H2O2 and made the interaction level of 14-3-3 protein and PHOT was higher than that of WT, and then the blue light signal transduction was better, which can promote the interaction of 14-3-3 protein and plasma membrane H+-ATPase and the activity of plasma membrane -ATPase and hydrogen pump, then increase the stomatal opening and conductance.3. Use of furniture in the pollution of low concentration gas HCHO treated WT and C1, C3 transgenic tobacco, in order to investigate that the effect of transgenic plant and WT response to HCHO stress when it was put in the environment of HCHO pollution for three days. The results are similar with the high concentration treatment that the stomatal opening and conductance of transgenic tobacco were higher than that of WT, and the oxidative damage index include H2O2, MDA and PC are less than WT, the number of guard cells which accumulation of H2O2 in transgenic tobacco is less than the WT tobacco. Immune coprecipitation and Western analysis show that the interaction level of 14-3-3 protein and 14-3-3 protein with plasma membrane H+-ATPase of transgenic tobacco were all higher than the WT, and the activity of plasma membrane H+-ATPase and hydrogen pump were higher than the WT. These results suggested that reduced accumulation of H2O2 in the leaves under gas HCHO stress, and then enhanced the interaction of 14-3-3 protein with plasma membrane H+-ATPase which activated the activity of plasma membrane H+-ATPase and hydrogen pump, which may be the important mechanism of over expression of CAT enhanced the stomatal movement and HCHO absorption of transgenic tobacco under gas HCHO stress.