Functional Analysis Of Formaldehyde Metabolism-related Gene Anfaldh In Transgenic Plant

Formaldehyde, one of the major indoor environmental pollutants, can cause human acute or chronic poisoning. Removal of formaldehyde by means of plant absorption is considered to be the most simple, natural, and environmentally friendly approach, although the capacity of ornamental plant to metabolize formaldehyde is far less than that human demands. By genetic engineering methods, the present study intends to enhance ability of tobacco plants removing formaldehyde by overexpressing of a glutathione-dependent formaldehyde(Anfaldh) gene from Aspergillus nomius SGFA1. The main contents are as follows:1. Construction of the plant expression vector pBI121-Anfaldh, and transformation of tobacco plant by Agrobacterium-mediated method.Plant expression vector pBI121-Anfaldh was constructed. Tobacco transformation was performed by Agrobacterium mediated method. A total of 50 kanamycin-resistant transgenic lines were obtained, and 27 lines were positive in PCR assay.2. Molecular identification of transgenic plantsTransgenic lines were selected for further qPCR analysis. The transgenic lines showed a range of 3.6-14 fold relative transcription levels of the Anfaldh gene. Three transgenic lines(designated as Anfaldh4, Anfaldh40, Anfaldh130) displayed higher transcriptional levels, whereas the Anfaldh4 is the highest on transcriptional level.3. FALDH enzyme activity assaysThe enzyme activity was determined spectrophotometrically by monitoring the production of NADPH at 340 nm. The FALDH specific enzyme activity in the extracts of Anfaldh4, Anfaldh40, Anfaldh130 and WT lines were 0.429, 0.392, 0.345, and 0.195 U mg-1 protein, respectively. Compared to WT, the 1.8-2.2-fold higher FALDH-specific enzyme activity in transgenic lines, indicated that the FALDH enzyme is active in transgenic tobacco.4. Phenotypic test of transgenic plantsIn the medium containing formaldehyde, wild-type tobacco growth was inhibited, and the transgenic plants can normal growth. Moreover, after gaseous formaldehyde treatment, the transgenic plants showed better growth than the WT plants. The leaves of transgenic lines kept green and healthy, while leaves of WT became damaged under HCHO strss. This suggest that the formaldehyde-degradation ability of overexpression plants is stronger than the WT plants.5. Physiological analysis on transgenic plantsUnder the formaldehyde stress, the MDA content increased significantly, but the transgenic lines were significantly lower than the WT plants. Chlorophyll、Soluble suger and Soluble protein content were significantly higher than WT plants. These findings indicates that overexpression plants confered a high tolerance to exogenous formaldehyde.6. Exploring on the formaldehyde-metabolize ability of transgenic tobaccoIn 2 mM formaldehyde solution, the uptake rate of the transgenic plants for aqueous HCHO was examined. The transgenic plants showed a higher uptake rate than the WT plants. After 72 h treatment, 93% of the total HCHO was absorbed in Anfaldh4, while 62% of the total HCHO remianed in the WT samples. Under the gas formaldehyde treatment, the absorbtion of formaldehyde in the chambers with WT plants exhibited the lowest value, showing that gaseous formaldehyde was effectively metabolized by the FALDH transformants.