Studies on chloroplast transformation of glyALB gene into cabbage and ectABC gene into rice
| Autor: | Ching-Chia Chen, 陳靖佳 |
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| Rok vydání: | 2016 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 104 Cabbage (Brassica oleracea L. var. capitata L.) and rice (Oryza sativa L.) are the important crops grown worldwide, and also has been the most widely cultivated crops in Taiwan. Traditional breeding methods have been making remarkable progress in the yield and quality of cabbage and rice over the past fifty years; however, the traditional approaches have been limited by genetic resources. Currently, biotechnological advances in gene transformation technology have opened new avenues to accelerate crop improvement. Bacteriocins are bacterially produced short-chain peptides that are active against closely related bacteria species. Two structural genes, glyA and glyB, encoding enzymes catalyzing Glycinecin A biosynthesis had been isolated and characterized from phytopathogenic bacteria Xanthomonas campestris pv. glycines by Dr. Ming-Te Yang’s laboratory, Institute of Molecular Biology, NCHU. A higher level of bactericidal activity was found in chimeric Glycinecin A protein, GlyA-linker-GlyB (GlyALB) that has 15 residues of (Gly4Ser)3 as linker to fuse GlyA and GlyB, than native Glycinecin A purified from X. campestris pv. glycine. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid), is a widespread cyclic amino acid synthesized by both halotolerant and halophile bacteria. Ectoine acts in microorganisms as an osmotic compatible solutes against halostress. Besides functioning as osmoprotectants, ectoine was shown to protect biomolecules such as proteins, nucleic acids, biomembranes, and whole cells against denaturation caused by heating, freezing, desiccation, or chemical agents. Overexpression of genes encoding the enzymes involved in the ectoine biosynthesis, ectA, ectB, and ectC, resulted in tolerance to salt stress in transgenic tobacco and tomato had been reported. Genes ectA, ectB, and ectC, had been cloned and characterized from moderate halophile Halomonas magadiensis by Dr. Ming-Te Yang’s laboratory. In this study, we proposed to engineering the GlyA and GlyB (GlyALB) genes into the chloroplast of cabbage, as well as ectA, ectB, and ectC (ectABC) genes involving the ectoine biosynthesis into the chloroplast of rice. The objectives of this study were to engineer transplastomic cabbage and rice with disease resistance and stress tolerance, respectively. Constructed chloroplast transformation vectors harboring the daao or aadA as selectable marker genes and GlyA and GlyB (GlyALB) genes or ectA, ectB, and ectC (ectABC) genes as target genes were transferred into cabbage and rice chloroplast, respectively, via biolistic bombardment. The regenerated plants were selected by D-alanine or and spectinomycin. The results of PCR analysis of putative transplastomic cabbage indicated that the transformed GlyA and GlyB (GlyALB) genes were present in the chloroplast genome of transplastomic cabbage plants. The results of PCR and RT-PCR analysis of putative transplastomic rice indicated that the transformed genes ectA, ectB, and ectC (ectABC) genes were present in the chloroplast genome of transplastomic rice plants, and its mRNAs were expressed. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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