| Sugarcane is the most important plant for sugar, and cane sugar occupies over90%of sugar production in China. However, over80%of the crop is planted in the hilly unpland area, where the rainfall is uneven and irrigation is not available, and drought has become one of the major limiting factors for sugarcane production in China. Breeding sugarcane varieties with strong drought tolerance and high water use efficiency is one of the most important strategies for sustainable development of the sugar industry. Due to the complex genetic background of sugarcane, however, crop modification through conventional hybrid breeding program is time costly and lowly efficient, other supplementary approaches including chemical regulation are necessary for improving the sugarcane drought resistance in practice. The aims of this research were to compare the physiological changes in leaves between the treatments of drought and drought plus foliar application of abscisic acid (ABA) under water stress conditions using sugarcane variety GT21, furthermore, to sieve the ABA responsive genes with microarray, and to clone the key gene involved in ABA biosynthesis and signal transduction.1. An experiment was set up to investigate the interrelationship between drought induced ABA biosynthesis and antioxidative defense system, and to confer the farther role of foliar application of ABA in imparting drought tolerance to the sugarcane plant. The results showed that drought treatment enhanced the ABA concentration in leaf but it was significantly higher in combined ABA treatment, suggesting its biosynthesis was triggered in leaf by the ABA application. The relative water content was dramatically decreased by drought treatment but it could maintain higher in ABA treatment. Both drought and ABA treatment could result in an increase in proline, soluble protein, auxin, H2O2and malondialdehyde (MDA) content but the plants applied with ABA were found to resist the accumulation of MDA and gibberellin. ABA application decreased the degradation of chlorophyll, counteracted, at least in part, the decrease in maximal PS Ⅱ efficiency(Fv/Fm), antennae efficiency of PS Ⅱ (Fv/Fm) and quantum efficiency of PSⅡ (PS2). Overproduction of H2O2in plants treated with water stress was followed by higher activities of catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and ascorbate peroxidase (APX), but this increase was further improved by the ABA treatment. The results clearly suggests that though the tolerant variety showed an enhanced protective system against drought conditions, the foliar application of ABA further improved its tolerance by continuously triggering the over expression of antioxidative defense system.2. Degenerated and specific primers were designed from highly homologous regions of NCED gene sequences of different crop plants taken from NCBI database, a full-length cDNA encoding NCED gene was isolated and characterized from leaves of Saccharum officinarum L.(So) using RT-PCR and RACE-PCR. The isolated full-length SoNCED (2521bp) with1827bp ORF, encoded a peptide of608amino acids. The calculated molecular weight of the protein was65.9kDa with isoelectric point of6.04. Conserved domains prediction indicated a chloroplast-targeting peptide located at N-terminus of SoNCED protein. Phylogenetic tree, constructed by Neighbor-joining method indicated that deduced protein sequences of SoNCED shared high identity with the NCEDs reported from Zea mays and Sorghum bicolor which all belong to the C4plants. Sequence alignment revealed that the basic secondary structure including a-helices,[3-strands, P-propeller and His residues coordinating catalytic sites of SoNCED were highly conserved as in the NCEDs from other plants. Tissue specific expression analysis using Quantitative real-time PCR (qRT-PCR) showed a significant increase in SoNCED mRNA level and its correlation with O2-production rate and ABA accumulation in leaves and roots of sugarcane variety GT21when exposed to water stress. Further, the stimulation of SoNCED mRNA level, O2-production rate and ABA content after exogenous application of ABA proved its involvement in pathways providing tolerance to drought stress. 3. An Agilent4×44K microarray was applied to investigate the gene differential expression under water stress and foliar application ABA. The results showed that the number of differentially expressed genes was higher in the drought plus ABA treatment than drought treatment only. Large part of the genes was first induced then depressed following the aggravated water stress in both treatments. Total231genes were identified as ABA responsive from ABA treated samples at3rd day. Compared to the samples taken at5th day,71of the aforementioned genes were specifically expressed at3rd day in ABA treated plants,72genes were co-expressed in all treatments and days,38genes were co-expressed in ABA treatment, and50genes were specifically expressed at5th day in drought treated plants. Annotation results done by BlastX showed that173of the231genes could be validated. Function categories indicated that the hypothetical proteins have taken up about10%of the annotated genes, while genes functioned in lipid metabolism, signal transduction, protein kinase and osmoregulation have taken up about9%separately. Genes related to protein and hormone metabolism have taken up about6%separately.5%of the genes are related to photosynthesis, carbohydrate metabolism and transport separately. The remaining genes take part in respiration, transcription, oxidative stress, nitrogen metabolism, cellular metabolism and so on. The differential expression patterns of nine candidate genes were chose confirmed by qRT-PCR and the results showed that the expression changes of these candidates were generally consistent with the microarray results.4. After annotation of the ABA responsive genes, one with highly homologous to the sucrose non-fermenting related protein kinase (SnRK2) gene SAPK3in Oryza sativa was selected for further analysis, and the cDNA full-length of this gene was amplified using RACE-PCR and named SoSnRK2.1. The isolated full-length of SoSnRK2.1was1385bp with1002bp ORF, encoding a peptide of333amino acids. Sequence analysis indicated that SoSnRK2.1showed high similarities with SnRK2from other plant species, and belong to the first subfamily when clustered with the SnRK2s taken from Arabidopsis thaliana using a Neighbor-joining method. The calculated molecular weight of the protein was37.8kDa with isoelectric point of5.54, and predicted as a hydrophilic protein without transmenbrane domains. There was a protein kinase conservative catalytic domain between the5-261amino acid sites, with11,3and8potential phosphorylation sites of serine, threonine and tyrosine separately, and the loop structure have taken the most part in the second structure prediction of SoSnRK2.1. Tissue specific expression analysis using qRT-PCR showed a significant increase in SoSnRK2.1mRNA level induced by foliar application of ABA and water stress, and correlated with ABA accumulation in leaves, which indicated the signal transduction function of this gene in leaves, while the expressions were suppressed with the accumulation of ABA content in roots, and this might be due to the redundancy of SnRK2genes in plants, but should be for further research. |
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