| Jatropha curcas L., grow as woody shrubs or small trees, has attracted extensive attention due to its high oil content in seeds and can be used as biodiesel resource.Nevertheless, because of its origin from the tropical regions, chilling and drought stress has become the main factor that limits it’s geographical distribution and yield.Proline and glycine betaine, acted as two kinds of the compatible organic osmotica synthesized in cells, play an most important roles of osmotic regulators in plants.Studies indicated that cold and drought-induced osmotic stress could lead to accumulate large amount of proline in plants, but the specific mechanism has been reported little. Another studies showed that abscisic acid(ABA) particpated in regulation of proline and betaine accumulation in plants under stresses. However, it’s mechanism is unclear in J. curcas seedlings. In this research, using J. curcas seedlings as experimental materials, the effect of ABA on accumulation proline and glycine betaine under chilling stress, and effect of osmotic stress on proline accumulation and metabolic pathways were investigated in J. Curcas seedlings.Furthermore, We cloned the CDS(Coding sequences) of stress-related genes,analysised their bioinformatics and primarily identificated their biology function. The main results are detailed as follows:1. Effect of exogenous abscisic acid on proline accumulation and metabolic pathways in Jatropha curcas seedlings under cold stressThe results showed that cold stress(5℃) led to a significant accumulation of proline in J. curcas seedlings. It also induced a rapid increase of activity of the key enzymes P5 CS and OAT of proline biosynthesis, and an up-regulation of JcP5 CS expression, and a decrease of the activity of key enzyme proline dehydrogenase(ProDH) of proline degradation. Treatment with 150 μmol/L ABA could enhance the accumulation of proline in J. curcas seedlings under cold stress. ABA also increased the activity of P5 CS, and improved the expression level of the JcP5 CS. Furthermore,the ABA treatment decreased the activity of ProDH in J. curcas seedlings under cold stress. These results showed that the ABA-induced proline accumulation in the J.curcas seedlings under cold stress might be a combined result of the activation of glutamate pathways of proline biosynthesis and inhibition of proline degradation pathway.2. Effect of exogenous abscisic acid on chilling tolerance and glycine betaine accumulation in Jatropha curcas seedlings under chilling ctressThe results showed that treatment with 150 μmol/L ABA could significantly increased survival rate and root vitality, decreased MDA content and electrolyte leakage in J. curcas seedlings under chilling stress. ABA also enhanced the accumulation of glycine betaine, increased the activity of BADH, and improved the expression level of the JcBADH. The results suggested that exogenous ABA treatment can enhance chilling tolerance in J. curcas seedlings, and glycine betaine might play a key role in the increased chilling tolerance.3. Effect of osmotic stress on proline accumulation and metabolic pathways in Jatropha curcas seedlingsIn this experiment, effect of osmotic stress generated by polyethylene glycol of different concentrations(PEG 6000) on proline accumulation and metabolic pathways in Jatropha curcas seedlings was investigated. The results showed that polyethylene glycol(PEG 6000) stress 4 d could lead to a significant accumulation of proline. PEG6000 concentration at 10%, proline content is relatively high, compared with the control, proline contents were increased by 212.1%. Furthermore, a rapid increase of the activities of the key enzymes P5 CS and OAT of proline biosynthesis by 114.3%and 77.0%, and a decrease of the activity of the key enzyme ProDH of proline degradation by 60.6%. The results of semi-quantitative RT-PCR revealed that 10%PEG 6000 stress activated the expression of JcP5 CS and JcOAT genes, and inhibited the expression of ProDH gene in J.curcas seedlings. These results showed that the osmotic stress-induced proline accumulation in the J. curcas seedlings might be a combined result of the activation of glutamate pathway and ornithine pathways of proline biosynthesis and inhibition of proline degradation pathway.4. Molecular cloning of JcICE1 and JcOAT from Jatropha curcas L.and recombinant yeast strain expression analysisFrom previous data of the transcriptome of Jatropha curcas L. in our laboratory,We use high fidelity Primestar enzyme to amplify the CDS of JcOAT and JcICE1 genes. The full length of our cloned JcICE1 CDS is 1626 bp, encoding a polypeptide of 542 amino acids, with theoretical molecular weight of 59.0 kDa and pI value of5.68, and typical bHLH and ACT motif. The full length of cloned JcOAT CDS is1425 bp, encoding a polypeptide of 474 amino acids, with theoretical molecular weight of 51.8 kDa and pI value of 6.4, and typical OAT_like and AAT_I motif. The JcOAT and JcICE1 genes were subcloned into the plasmid pYES2 to create the yeast expression vector pYES2-JcOAT/JcICE1. Through the colony dot-plating test and spectrometric analysis of yeast dynamic growth curve, Saccharomyces cerevisiae wild-type strain INVSc1,bearing the recombinant vector pYES2-JcOAT was found to demonstrate a notably better growth under osmotic stress(30%, 40%, 50% PEG) 1 d than the control strain of the empty vector pYES2, growth rate is also significantly increased. implicating the heterologously expressed JcOAT could also enhance the osmotic tolerance of S. Cerevisiae. After 1 d at the cold(-20 ℃) stress, the recombinant vector pYES2-JcICE1 yeast growth rate are also significantly better than the control strain of the empty vector pYES2, indicating that overexpressed the gene JcICE1 improves the yeast cold resistance ability.5. Molecular cloning of JcSP1 gene and it’s stress resistance analysis in transgenic tobacco plantsWe used genome DNA as template and then amplified the JcSP1 gene. The full length of our cloned JcSP1 CDS is 330 bp, encoding a polypeptide of 109 amino acids, with theoretical molecular weight of 12.266 KDa and pI value of 5.43, the domain belongs to Dabb superfamily, stress responsive a/b barrel domain. Then,subcloned into the plant expression vector pCAMBIA3301 plasmid by enzyme cutting, constructed plant vector pCAMBIA3301-JcSP1, and transformed it into LBA4404. Agrobacterium-mediated leaf disc transformation method to obtain transgenic T0-generation transgenic tobacco plants. By genomic PCR, RT-PCR identification method to get the JcSP1 transgenic tobacco plants. T1 generation seeds resistance analysis found that high temperature, cold, salt and osmotic stress,transgenic seeds were significantly better than the wild type. The results showed that transferred the JcSP1 increased the stress resistance capability of transgenic tobacco. |
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