Options
Isolation and genetic engineering of stilbene synthase gene encoding for natural resveratrol production in red-leaf vegetable
Author
Ng, Irene
Supervisor
Chia, Tet Fatt
Abstract
Resveratrol(3,4',5-trihydroxystilbene), a stilbenoid with various biological activities such as being an antioxidant, anti-cancer compound, anti-platelet aggregation compound, phytoalexins and phytoestrogen. It is synthesized in a one-step reaction fiom one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA by stilbene synthase (STS). These precursors are found abundantly in plants that contain high level of anthocyanins.
The aims of this project are (1) to isolate and characterize the STS genes hm Vitis vinifera CV. Red Flame; (2) to determine the best cultivar of Luctuca sativa (red lettuce) to use for transformation; (3) to test the functionality of the isolated V. vinifera CV. Red Flame STS genes by transforming the genes into L. sativa (red lettuce), so as to determine the STS with the strongest activity; and, (4) to determine the effect of over-expression of STS on the anthocyanins level in L. sativa (red lettuce).
Partial characterization showed that there are at kast 6 STS genes present in the genome of V. vinifera CV. Red Flame. Full characterization of 4 full-length STS genes showed 98 % homology to VRSTS (G14), 99 % homology to pSV21 (G17), 97 % homology to pSV25 (Rl) and 98 % homology to pSV25 (R65).
Due to high anthocyanin level and high re-generation abiiiy, L. sativa CV. Red Salad Bowl were chosen to be used as the plant material for transformation of the isolated V. vinifera CV. Red Flame STS genes.
4 isolated full-length STS genes in expression vector (pBIG14, pBIG17, pBIRl and pBIR65) driven by CaMV 35s promoter were transformed into L. sativa CV. Red Salad Bowl and Nicotiana tabacum CV. Xanthi. For L. sativa CV. Red Salad Bowl 9 lies (for pBIG14), 5 lines (for pBIG17), 3 lines (for pBIR1) and 1 line (for pBIR65) were obtained. As for N. tabacum CV. Xanthi, 3 lines (for pBIG14), 5 lines (for pBIG17), 5 lines (for pBIR1) and 4 lines (for pBIR65) were obtained. The best line was chosen for the 4 constructs for both L. sativa CV. Red Salad Bowl and N. tabacum CV. Xanthi.
A11 4 V. vinifera CV. Red Flame STS genes (G14, G17, R1 and R65) were shown to integrate into the genome of L. sativa CV. Red Salad Bowl and N. tabacum CV. Xanthi and 1.3 kb STS transcripts were obtained for all the 4 V. vinifera CV. Red Flame STS genes. In transgenic N. tabacum CV. Xantbi, G14 was found to have a lesser tramcript level as compared to G17, R1 and R65. But, in transgenic L. sarivo CV. Red Salad Bowl G14 was found to have the most transcripts among the 4 V. vinifera CV. Red Flame STS genes.
With the presence of more precursors in L. sativa CV. Red Salad Bowl it was found that the resveratrol amount increased on the average of l l-fold for G14 (0.60 μg/g fresh weight), G17 (4.80 μg/g fresh weight) and R1 (0.94 μg/g fresh weight). With the exception of R65 (0.4 μg/g fresh weight) which was about the same as N. tabacum CV. Xanthi (G14 = 0.09 μg/g fresh weight, G17 = 0.27 μg/g fresh weight, R1 = 0.15 μg/g fresh weight and R65 = 0.36 μg/g fresh weight). This increased in resveratrol amount did cause the intensity of the red color of the transgenic L. sativa CV. Red Salad Bowl to be lower than the wild-type L. sativa CV. Red Salad Bowl.
In conclusion, the 4 V. vinifera CV. Red Flame STS genes were successfully isolated and transformed into both L. sativa CV. Red Salad Bowl and N. tabacum CV. Xanthi. There is no conclusive evidence to show the strongest activity STS genes among the 4 genes. Stronger constitutive promoter such as actin promoter or double promoters or the use of viral omega sequence for more efficient translation are therefore needed in order to cause a significant change in the anthocyanins level of L. sativa CV. Red Salad Bowl.
Since we had shown that the yield of resveratrol increased in red-leaf plant, in this case, red lettuce (L. sativa CV. Red Salad Bowl), this system can also be used in other colored plants and hits. This novel invention has the potential in paving the way for new generation of vegetable or fruits nutriceuticals that have chemo-preventive ability against cancer, cardiovascular and other diseases. Hence, we had filed a patent for this novel invention (Singapore Patent Application No.: 200006741 -3).
The aims of this project are (1) to isolate and characterize the STS genes hm Vitis vinifera CV. Red Flame; (2) to determine the best cultivar of Luctuca sativa (red lettuce) to use for transformation; (3) to test the functionality of the isolated V. vinifera CV. Red Flame STS genes by transforming the genes into L. sativa (red lettuce), so as to determine the STS with the strongest activity; and, (4) to determine the effect of over-expression of STS on the anthocyanins level in L. sativa (red lettuce).
Partial characterization showed that there are at kast 6 STS genes present in the genome of V. vinifera CV. Red Flame. Full characterization of 4 full-length STS genes showed 98 % homology to VRSTS (G14), 99 % homology to pSV21 (G17), 97 % homology to pSV25 (Rl) and 98 % homology to pSV25 (R65).
Due to high anthocyanin level and high re-generation abiiiy, L. sativa CV. Red Salad Bowl were chosen to be used as the plant material for transformation of the isolated V. vinifera CV. Red Flame STS genes.
4 isolated full-length STS genes in expression vector (pBIG14, pBIG17, pBIRl and pBIR65) driven by CaMV 35s promoter were transformed into L. sativa CV. Red Salad Bowl and Nicotiana tabacum CV. Xanthi. For L. sativa CV. Red Salad Bowl 9 lies (for pBIG14), 5 lines (for pBIG17), 3 lines (for pBIR1) and 1 line (for pBIR65) were obtained. As for N. tabacum CV. Xanthi, 3 lines (for pBIG14), 5 lines (for pBIG17), 5 lines (for pBIR1) and 4 lines (for pBIR65) were obtained. The best line was chosen for the 4 constructs for both L. sativa CV. Red Salad Bowl and N. tabacum CV. Xanthi.
A11 4 V. vinifera CV. Red Flame STS genes (G14, G17, R1 and R65) were shown to integrate into the genome of L. sativa CV. Red Salad Bowl and N. tabacum CV. Xanthi and 1.3 kb STS transcripts were obtained for all the 4 V. vinifera CV. Red Flame STS genes. In transgenic N. tabacum CV. Xantbi, G14 was found to have a lesser tramcript level as compared to G17, R1 and R65. But, in transgenic L. sarivo CV. Red Salad Bowl G14 was found to have the most transcripts among the 4 V. vinifera CV. Red Flame STS genes.
With the presence of more precursors in L. sativa CV. Red Salad Bowl it was found that the resveratrol amount increased on the average of l l-fold for G14 (0.60 μg/g fresh weight), G17 (4.80 μg/g fresh weight) and R1 (0.94 μg/g fresh weight). With the exception of R65 (0.4 μg/g fresh weight) which was about the same as N. tabacum CV. Xanthi (G14 = 0.09 μg/g fresh weight, G17 = 0.27 μg/g fresh weight, R1 = 0.15 μg/g fresh weight and R65 = 0.36 μg/g fresh weight). This increased in resveratrol amount did cause the intensity of the red color of the transgenic L. sativa CV. Red Salad Bowl to be lower than the wild-type L. sativa CV. Red Salad Bowl.
In conclusion, the 4 V. vinifera CV. Red Flame STS genes were successfully isolated and transformed into both L. sativa CV. Red Salad Bowl and N. tabacum CV. Xanthi. There is no conclusive evidence to show the strongest activity STS genes among the 4 genes. Stronger constitutive promoter such as actin promoter or double promoters or the use of viral omega sequence for more efficient translation are therefore needed in order to cause a significant change in the anthocyanins level of L. sativa CV. Red Salad Bowl.
Since we had shown that the yield of resveratrol increased in red-leaf plant, in this case, red lettuce (L. sativa CV. Red Salad Bowl), this system can also be used in other colored plants and hits. This novel invention has the potential in paving the way for new generation of vegetable or fruits nutriceuticals that have chemo-preventive ability against cancer, cardiovascular and other diseases. Hence, we had filed a patent for this novel invention (Singapore Patent Application No.: 200006741 -3).
Date Issued
2000
Call Number
QH442 Ng
Date Submitted
2000