| Terpenoids are a kind of main secondary metabolites in plants, and closely related to plant growth and development and the adaptation to the environment. Metabolic engineering has been one of the most potential technologies to study the metabolism and regulation of terpenoids. In the past few decades, People focused on studying the terpenoids defensive effect on plants. Nowadays, more and more research focus on the compartmentation of key enzyme of the process of terpenoids metabolism. It aims to study the influence of terpenoids metabolism on plant growth and development through changing the location of key enzyme. Apple α-Farnesene synthase (α-AFS) is the last enzyme in the synthesis of sesquiterpene α-farneesene. It plays an important role in the the synthesis of α-farnesene. α-AFS was proved to be located in cytoplasm via the prediction of software and research. CaMV35S-AFS gene was first transformed into tobacco, and got markedly phenotype of the homozygote of transgenic plants. In this sthdy, Rubisco-plasmid/mitochondria targeting-AFS fusion gene expression vector was constructed to overexpress a-AFS gene in chloroplast and mitochondria in tobacco cells, respectively, and studied the effects of main enzymes expression and terpenes metabolism profile which included a-farnesene in the isoprenoid metabolic pathway. The aim was to futher investigate the a-AFS expression in different parts of transgenic plants. Meanwhile, we had a preliminary study on the physiological characteristics and plant resistance of transgenic plants. The main results are as follow:(1) We isolated the AFS, promoter of Rubisco small subunit gene (Ru), chloroplast signal peptide (Pla), mitochondria transit peptide (Mit) from white winter pearmain peels, Chrysanthemum morifolium, Arabidopsis thalianas and Yeast, respectively.(2) The prediction result of PlantCARE showed that the sequence of Ru had multiple TATAbox, CAATbox and some hormone response element. It had typical characteristics of strong promoter. We had a prediction of chloroplast signal peptide and mitochondria transit peptide by SignalP4.1Server. Both of them had the function of signal peptide.(3) We constructed the expression vector pBI121+Ru+Pla/Mit-GFP and then transformed into Arabidopsis thaliana or transient transformed into onion epidermal cells. Results showed that Pla and Mit could transport GFP correctly into chloroplast or mitochondria, respectively. At the same time, it also proved that Ru could activate gene expression.(4) pBI121+Ru+P1a/Mit+AFS expression vector was constructed and transformed into tobacco. The transgenic lines were verified by PCR and qRT-PCR. We generated transgenic plants contained pBI121+Ru+Pla/Mit+AFS.(5) In chloroplast over-expressing transgenic plants, NtHMGR2and NtFPS expression were significantly higher than that of the wild type from4leaves to floral bud period. But in florescence, the expression of NtHMGR2in WT was higher than transgenic plants. NtFPS expression difference was not significant between them. Transgenic plants exhibited higher NtDXS and NtDXR expression level from4to12leaves stage, and had equally expression level at other two stages. In mitochondria over-expressing transgenic plants, the expression of NtHMGR2was higher than that of WT at the floral bud period. From4leaves to floral bud period, the NtFPS expression level in transgenic plants was higher. The expression of NtDXS and NtDXR was higher than WT at4and8leaves stage. However, it was lower than WT in florescence.(6) The chlorophyll content of the chloroplast over-expressing transgenic plants was higher than that of WT at the whole growth and development period. However, carotenoids content of transgenic plants was higher than that of WT from4to12leaves stage. At the floral bud period, carotenoids content in WT was higher. And it had no obvious difference between them in florescence. The chlorophyll and carotenoids content of the mitochondria over-expressing transgenic plants were higher than that of WT at the whole growth and development period.(7) The space expression of a-AFS had nothing to do with the localization of a-AFS. The expression of a-AFS in leaf was the highest, which was far higher than that of root, stem and flower.(8) We did not detect the a-farnesene in leaf and flower of transgenic plants. But there were different volatile substances in transgenic and WT plants. In flowers, contents of P-linalool and P-caryophyllene were different. The P-linalool content of WT was lower than that of transgenic plants and β-caryophyllene content was higher than that of transgenic plants. The leaf alcohol content in transgenic plants leaf was higher. The content of P-caryophyllene in leaf was lower than that of flower and the difference of β-caryophyllene content in plants leaf was not significant.(9) The insects feeding rate of transgenic plants was far higher than that of WT and the defensive ability to insect lowered.(10) Over-expressing a-AFS in tobacco chloroplast enhanced the resistant ability to high temperature, drought and oxidation stress. However, over-expressing a-AFS in tobacco mitochondria reduced the resistant ability. |
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