| Drought is the main factor that restricts the sugarcane productivity in china. The breeding and selection for new sugarcane varieties with new high yield, high sucrose, drought-resistance and wide regional adaptability are the effective way to improve the cane productivity under seasonal drought in China. Transgenic sugarcane with high drought resistance and water use efficiency gene is a new methodology to improve the sugarcane drought resistance. Drought resistance of sugarcane includes the drought tolerance and the ability to recover the growth after stress relief. Therefore, studies on the physiological and molecular mechanisms of sugarcane in response to drought stress and re-watering will aid in the understanding of the genetic improvement involved in drought resistance.Sugarcane is C4crop with high photosynthetic efficiency and many advantageous characteristics, such as drought resistance, waterlogging tolerance, barren tolerance, disease resistance, high biomass and so on, which is an important genetic resource for crop improvement, In order to understand the physiological and molecular mechanisms of sugarcane in response to water stress, and find drought-tolerant genes, the strongly drought resistant sugarcane variety GT11, was used as the plant material. The physiological characteristics including the osmotic adjustment, protective enzyme activities, physiological metabolisms, and endogenous hormones, etc. were analyzed to investigate the physiological response to drought stress and re-watering in sugarcane. The differential genes induced under drought stress and re-watering, were isolated and screened using two methods of cDNA-AFLP and cDNA-SCoT. The full-length cDNAs from a part of specific differential genes were obtained by cloning, which could provide gene resources for genetic improvement of the crop drought resistance. The main research contents and results are as follows:1. Sugarcane variety, GT11showed the physiological reposes to water stress including osmotic adjustment, protective enzyme activities, substance and endogenous hormones metabolism, etc. during elongating stage, which indicated the strong ability of GT11to adapt to water stress.(1) Under drought stress, osmotic adjustment substances, soluble sugar and proline were largely accumulated and P5CS activity was increased, while the soluble protein content and ProDH activity were decreased in sugarcane leaves. After re-watering, the soluble sugar content in leaves was decreased sharply and then increased to the level closed to control, proline content and P5CS activity were decreased rapidly, on the contrary, the soluble protein and the ProDH activity increased fast.(2) Under drought stress, the contents of MDA and the superoxide anion were increased and the activities of protection enzymes including peroxidase, catalase, superoxide dismutase and glutathione reductase were increased in different levels. After re-watering, the contents of MDA and the superoxide anion were decreased rapidly, and the activities of peroxidase, superoxide dismutase and glutathione reductase were also decreased, then increased, and the catalase activity was first decreased and then increased.(3) Under drought stress, abscisic acid content was highly accumulated as the drought intensifying, and polyamine oxidase activity was enhanced in a certain range. After re-watering, abscisic acid content was increased first and then decreased, and polyamine oxidase activity was decreased rapidly.2. Four hundred and six TDFs were isolated through cDNA-AFLP analysis. One hundred and sixty-seven among them were verified as positive by reverse Northern blotting. Twenty-seven TDFs were cloned, sequenced, BLAST analyzed and classified by functional analysis as seven categories, including energy metabolism, transcription regulation, protein binding, environmental interaction and other functional genes.3. There were316TDFs isolated from the cDNA-SCoT analysis for the gene differential expression induced by water stress. Among them180of positive TDFs were obtained using reverse Northern blotting excluding the false positives. One hundred seventy seven of positive TDFs were cloned, sequenced, and the BLAST analyzed. There were107TDFs showed high similarity to the known genes in NCBI database,28TDFs showed high similarity genes to the hypothetical protein with unknown functions in NCBI database. Total one hundred thirty five TDFS were divide into fourteen catagories by functional analysis, which involved in metabolism, energy, transport, communication and signal transduction, cell cycle and DNA processing, transcription, protein synthesis, protein fate, binding function protein, transposable elements, viral and plasmid proteins, cell fate, defence, and unclassified proteins.4. Twelve TDFs isolated with cDNA-SCoT were selected and verlidated by semi-quantitative RT-PCR analysis. The results showed that the expressions of the twelve genes were induced by drought stress and re-watering. The expression patterns among of those genes could be classified into three types by functional analysis. Four genes are induced by drought stress, as the drought resistance gene; seven genes were up-regulated by re-watering, as growth regulatory genes; one gene was up-regulated by both droutht and re-watering, as a dual functional gene.5. Five differentially expressed genes were cloned by homology cloning method with the first cDNA strain from sugarcane variety GT11. The genes included ATP synthase CFO subunit IV (Sc-atpl), sucrose transporter1(Sc-SUTl), heat shock protein70(Sc-HSP70), S-adenosylmethionine decarboylase3(Sc-SAMDC3), and tubulin alpha-3chain (Sc-Tuba3). The sequences of nucleotide acids and amino acids, domain, hydrophobicity, multiple alignment and phylogenetic tress of these genes were analysised. The results showed that five genes from sugarcane had a high homology with other gramineous crops in NCBI database, such as maize and sorghum. Sc-atpl, Sc-Tuba3, Sc-HSP70and Sc-SAMDC3were first cloned from sugarcane. |
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