| Gibberellin(GA) is a kind of endogenous hormones in plants, and plays an important role in regulation in entire life cycle of plant. The GA-20 oxidase and GA-2 oxidase are key enzymes of gibberellic acid synthesis pathway in the third stage; they are encoded by multi-gene families, and participants in the regulation of gibberellin level. GA-20 oxidase can promote the synthesis of activity gibberellin, this pattern belongs to the positive regulation. While GA-2 oxidase translates active gibberellin into inactive gibberellin, this pattern belongs to the negative regulation. So the expression level of GA20 ox gene and GA2 ox gene affect the the synthesis of active GA in plants and ultimately affect the growth and development of plant.In this study, in order to further understand the relationship between the plant height and gene expression of key enzyme in GA synthesis, the plant height mutant potato was used as material first; the transgene copy number was detected by using quantitative real-time PCR. And then the gene expression of key enzyme in GA synthesis in different period and different tissue of potato plant was measured. The GA2ox1 gene and GA20ox1 gene were cloned and their sequence characteristics were analyzed. The effects of overexpression on potato plants were analyzed by agrobacterium mediated genetic transformation, and from the molecular level to elucidate the possible mechanism of two genes. This study the main results were as follows:1 Using quantitative real-time PCR method to estimate the foreign gene copy number in transgenic potato. The results showe that, two copies or more than two copies of T-DNA insertion is easy to cause some traits change in potato plant.2 The expression patterns of the key genes in the gibberellin synthesis pathway in the potato dwarf mutant M4P9 were detected using quantitative real-time PCR. These genes showed expression patterns that varied based on growth stage and tissue type. The overexpression of CPS1 gene provides ample endogenous hormone for plant growth in seedling stage. GA20ox1 gene changes obviously in the stem, that may be associated with tuber starch accumulation. The overexpression of GA2ox1 gene inhibits the synthesis of endogenous GA, which is considered to be one of the factors prompting the dwarf mutant of potato plant. Spraying GA3 can inhibit the GA synthesis in plant. The main performance is the gene expression down-regulation of gibberellin related enzyme.3 StGA2ox1 was cloned, and its CDS is 1005 bp, encoding 334 amino acids. The results of secondary structure showed that, the protein include alpha helix for 34.43%, random coil for 34.13%, extended strand for 22.16% and beat turn for 9.28%. Homology alignment results show that, StGA2ox1 has higher homology with SlGA2ox2, NtGA2ox1, NtGA2ox2 and NtGA2ox3.4 StGA20ox1 was cloned, and its CDS is 1137 bp, encoding 378 amino acids. The results of secondary structure showed that, the protein include alpha helix for 39.68%, random coil for 33.33%, extended strand for 19.58% and beat turn for 7.41%. Homology alignment results show that, StGA20ox1 has higher homology with SlGA20ox1, Nt GA20ox1 and SdGA20ox1.5 Build a new plasmid vectors pCAEZ1383, successfully. We constructed two plant expression vectors, 1383-GA2ox1 and 1383-GA20ox1. After the genetic transformation and PCR detection, seven transgenic plants of StGA20ox1 were identified and one transgenic plants of StGA2ox1 was identified.6 Using quantitative real-time PCR methods to detect the gene expression of StGA20ox1 and St GA2ox1 in transgenic lines of potato, the results showed that, the expression levels of StGA20ox1 and StGA2ox1 were up-regulation. And excessive expression of StGA20ox1 gene can promote the plant height growth and root elongation on potato plants.7 Semi-quantitative PCR results showed that, overexpression of StGA2ox1 can increase the resistance of transgene plant to abiotic stress in different degree. Under drought stress, transgenic plant has a relatively higher content of chlorophyll, free proline and relative leaf water. Overexpression of StGA20ox1 gene has little influence on potato response to abiotic stress. |
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