Construction and Transformation of Sulforaphane Biosynthetic Related Genes of Broccoli
| Autor: | Yung-Shen Lin, 林泳伸 |
|---|---|
| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 105 Cruciferous vegetables, such as broccoli, cabbage, and cauliflower, are rich sources of sulforaphane. Sulforaphane have been found to inhibit proliferation of cancer cells, prevent cancer cells to develop, and anti-metastasis of cancer cells. Sulforaphane is an isothiocyanate which is produced from glucosinolates in myrosinase-catalyzed hydrolysis. Myrosinase (MYR) is physically separated from glucosinolates in intact plant cells. Production of sulforaphane is upregulated by the MYR and ESM (epithiospecifier modifier) and downregulated by ESP (epithiospecifier protein). The complicated separation and purification procedure result in low extraction efficiency and extremely high price for the sulforaphane, which hampers a wide application in pharmaceutical science. Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetable crops grown worldwide, and also has been the most widely cultivated leafy vegetables in Taiwan. Broccoli (Brassica oleracea var. italica Planck) is well established as the best anti-cancer vegetable among the cruciferous vegetables. Using transgenic crops as bioreactors to produce industrial, feed and fodder additives, and pharmaceutical proteins become a new way for increasing economic values of crops and may solve the problems we are facing. By using the art of genetic engineering, we attempt to modify the glucosinolate-myrosinase substrate-enzyme system. Increasing MYR and ESM expression and knockdown the expression of ESP are achieved by chloroplast gene transformation and RNAi manipulation, respectively. Thus, overexpression of MYR and ESM in chloroplast and suppression of ESP expression, thorough breakdown of modified broccoli and cabbage will enhance the production of sulforaphane. In this study, Brassica chloroplast transformation vectors harboring the MYR and ESM genes were constructed. Vectors harboring antisense--oriented ESP gene (ESPi) were also constructed for Agrobacterium-mediated transformation. Six Brassica chloroplast transformation vectors had been constructed, namely pMT91t-Esm-A, pMT91t-Myr-A, pMT91t-Esm-Myr-A, pMT91t-Esm-GA, pMT91t-Myr-GA and pMT91t-Esm-Myr-GA. Two RNAi constructs for ESP gene has been obtained, namely, p1304-Esp-IN-GH and p1304-Esp-IN-GD. Brassica chloroplast transformation vectors, pMT91t-Esm-Myr-GA (harboring Myr and Esm gene), was transferred into the hypocotyls of ''K-Y cross'' cabbage and ''Green King'' broccoli via biolistic bombardment. Transformed plantlets are selected by 10 ppm spectinomycin. The results of PCR, RT-PCR, and qrt-RT-RCR analysis indicated that the transformed genes are present in the chloroplast genome of transplastomic plants, and expressed its mRNA. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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