Analysis of Infrared Spectral Features and Identification of Al-responsive Genes in Small Black Soybean with Aluminum Stress
|School||Kunming University of Science and Technology|
|Keywords||Fourier transform infrared spectroscopy (FTIR) suppression subtractive hybridization cDNA library Al-responsive genes small black soybean|
Currently, acidic soil accounts for 40% of the world arable lands. Aluminum (Al) toxicity is one of the major factors limiting the growth of plants in acidic soils. Along with the increase of population and the reduction of arable lands, improvement and utilization of acid soils have received much attention. Lime and alkaline fertilizer are used to reduce soil acidity in agriculture, but this method costs high and could not fundamentally solve problems. From the long-term benefits, application of genetic engineering means, breeding and utilization of crop resistance to aluminum is the most economic, effective and pollution-free fundamental measure. Yunnan is a productive region of small black soybeans. There is mainly red soil in Yunnan region, and soil acidification is serious increasingly by the influence of intensive management in modern agriculture. Therefore, it is necessary to study on aluminum detoxification mechanism in small black soybean. In this research, we explore aluminum detoxification mechanism of the small black soybeans through determining the physiological indexes, analysing the Fourier infrared spectral features and constructing the forward suppression subtractive hybridization (SSH) cDNA library of small black soyabean under aluminum stress. Main results were as follows:1. With the 50μmol·L-1 Al treatment, the relative root elongation of Yunnan small black soybeans were obviously higher than Medicago sativa. Root dyeing experiments also indicated that the Al tolerance of Yunnan small black soybeans was relatively stronger than Medicago sativa. The contents of soluble protein, hydrogen peroxide, soluble sugar and malondialdehyde were increased intensively for small black soybeans roots and leaves with the aluminum stress. There were fluctuation of the contents with different time treatment of aluminum stress, but some of them also have ascending trend. The activities of superoxide dismutase, catalase and peroxidase decline basically with the increase of aluminum stress and the extension of time. The activity of superoxide dismutase appears rising trend with the extension of aluminum stress time.2. The Fourier transform infrared spectroscopy (FTIR) technical analysis showed that there were differenttions among the FTIR spectraes of root, stem and leave of Yunna small black beans under different concentrations of aluminum stress. The characteristic peaks of sugar and methylation were increased with the increase of the concentration of aluminum. When the aluminium concentration increased to 100mol·L-1, Al toxicity was strengthened and the peak was declined. The change of peaks in roots stems and leaves of small black soybeans had different trends with different time treatment of aluminum stress. Under short time aluminum stimuli, the amount of organic compounds such as amino acid, carbohydrate, lipid, etc would be increased, which would play an important role in osmoregulation. With extension of the aluminum stimulation, synthesis and transportation of the organic compounds were hampered and the contents were declined in different degree.3. SSH method was adopted to construct a forward SSH-cDNA library for small black soybean, in this way, it is easy to analysize the genes induced by Al stress. Sequence analysis gets 159 ESTs, among which 102 ESTs are known function genes, including 26% related to metabolism,25% related to defense, stress and cell death,14% related to protein synthesis,13% related to signal transduction and transcription,8% related to transporter, 9% related to protein destination/storage,3% related to cell structure and cell growth, the least related to energy is about 2%. Gene unknown function needed to be further identified. The information obtained from the SSH library established an important foundation for the Al detoxification mechanism study in small black soybean at molecular level.4. Expression profiling of the 10 genes was analysed with RT-PCR, which confirmed that UDP-glucose 4-epimerase, Quinone oxidoreductase, Mn superoxide dismutase, Cu/Zn superoxide dismutase, p-type H+-ATPase, glutathione S-transferase, peroxisomal membrane protein, mitogen activated protein kinase, cytochrome P450, class I chitinase were up-regulated. This further illustrated the reliability of the library.