Antimicrobial activity of stilbenes compounds isolated from transgenic
The rate of increase of food crop production has decreased due different factors such as global climate change, alteration in use of land, pests, disease, salinity and drought. Food production was said to be inadequate for the increasing world population (WHO, 1996a). Therefore, it is essential to increase food production and distribution in order to meet its demand and free from hunger. Furthermore, transgenic plants can enhance yields, harvesting of crops, reduce dependency on chemical insecticides. Production of transgenic plants can address the global problems such as climate change, deficiency of food and nutrition. Development of transgenic plant involves manipulation or transfer of genes from other organisms which may improve yield, quality, herbicides, and pest or diseases resistant or environmental conditions, increased agricultural productivity and better quality foods. Modification of genetic constitute of plant by inserting transgene enhances nutritional composition of the foods and improve human health and minimizes the use of pesticides and insecticides.
A number of transgenic crop plants has been produced from a variety of crop plants to date with enhanced agronomic characteristics, for example, transgenic tomatoes with improved shelf-life, transgenic fruits and vegetables with delayed ripening time and increased length of storage. Moreover, pest and disease resistance crops have been produced, viz., papaya-ringspot-virus-resistant papaya (Gonsalves 1998), insect resistance cotton, transgenic rice plants that are resistant to rice yellow mottle virus (RYMV) (Pinto et al 1999), improved nutritional contents in the transgenic rice which exhibits an increased production of beta-carotene as a precursor to vitamin A (Ye et al 2000). In addition, technology of transgenic plant production can be used to produce vaccines and bioactive compounds in plants. For example, expression of anti-cancer antibody in rice resulted in production of vaccines against infectious disease from potato (Thanavala et al 1995).
Resveratrol is found in a limited number of unrelated plants and possess antifungal activity and induction in response to pathogen infection. Resveratrol is well known for its potent antioxidant activity and health-promoting effects, cardioprotection (Ignatowicz and Baer-Dubowska 2001) and reduction of cancer risk have also been observed (Jang et al. 1997; Cal et al. 2003).It can also exert neuroprotective effects by increasing heme oxygenase activity in the brain (Zhuang et al. 2003). The expression of RS transcripts has been associated with an increasedresistance to various fungal pathogens in transgenic tobacco (Hain et al. 1993) and tomato (Thomzik et al. 1997).
The purpose of this report is to better understand the role of transgene to improve the nutrition value of important crops and to evaluate the biological activity of secondary metabolic substances such as resveratrol, SOD, phenolic compounds in
2. Transformation of
R. glutinosa with RS gene
The peanut RS genomic DNA sequence, AhRS3 (GenBank Accession number, AF227963) a polypeptide of 389 amino acid residues, was cloned into the Xba I/Cla I sites of binary expression vector pGA643 under the CaMV35S promoter. This produced a recombinant AhRS3 expression plasmid, pMG-AhRS3.
Transgene-positive T0 lines were selected by PCR screening. The lines containing RS gene and
2.1. Scavenging of DPPH radicals and Inhibition of lipid peroxidation of transgenic
Free radical-scavenging activity was evaluated using trolox as standard antioxidants. The radical-scavenging activity was measured using the stable radical 1,1-diphenyl–2-picrylhydrazyl (DPPH) as previously described (Xing et al., 1996). Various concentrations of the extracts were added to 4 ml 0.004% methanol solution of DPPH. The mixture was shaken and left for 30 min at room temperature in the dark, and the absorbance was measured with a spectrophotometer at 517 nm. The radical-scavenging activity was expressed as a percentage of the absorption of DPPH in the presence and in the absence of the compound. Calculated IC50 values indicate the concentration of sample required to scavenge 50% of the DPPH radical. DPPH activity was calculated as
where, Ablank is the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound.
Inhibition of lipid peroxidation was determined by measuring thiobarbituric acid-reactive substance production (Buege and Aust 1978).
2.1.1. Measurement of photosynthesis rate
Stomatal conductance (
2.1.2. HPLC analysis
The HPLC analysis was applied using the modified method of Banwart et al. 1985. The mobile phase consisted of solvent A and B. Solvent A contained 98% water and 2% glacial acetic acid in 0.018M ammonium acetate. Solvent B was 70% solvent A and 30% organic solution, the latter being composed of 82% methanol, 16% n-butanol and 2% glacial acetic acid in 0.018M ammonium acetate. Following injection of 20µL of the sample, the flow rate of the mobile phases was maintained at 1mL min−1. A linear HPLC gradient was employed. The HPLC system consisted of a Young-Lin M930 liquid chromatograph pump and an M720 detector (Young-Lin Instruments Co., Ltd). The column for quantitative analysis was a YMC-Pack ODS-AM-303 (250×4.6mm I.D.), and the UV absorption was measured at 280 nm.
2.1.3. SOD activity
SOD activity of root
2.1.4. Paper disc diffusion assay
Bacterial pathogens and fungal strains were grown in liquid medium (micrococcus, nutrient, and YM media) for 20 h to a final concentration of 106–107 CFU/ml. Aliquots of 0.1 ml of the test microorganisms were spread over the surface of agar plates. Sterilized filter-paper discs (Whatman No. 1, 6 mm) were saturated with 50 µl of the methanol extract at 10,000 ppm and left to dry in a laminar flow cabinet. The soaked, dried discs were then placed in the middle of the plates and incubated for 24 h. Antimicrobial activity was measured as the diameter (mm) of the clear zone of growth inhibition. Negative controls were prepared using the same solvents employed to dissolve the plant extracts.
In order to evaluate morphological and agronomic performance of transgenic
2.2. Results and discussion
2.2.1. Biological activities of transgenic
184.108.40.206. Scavenging of DPPH radicals of transgenic
The free radical scavenging activities of non-transgenic control and transgenic R. glutinosa extracts, α-tocopherol, are presented in Fig.1. A solution of each extract at a concentration of 1.0 mg/ml was prepared. The activities of transgenic sample extracts were between 16.00 and 20.00 µg/ml at 1.0 mg/ml. Most of the transgenic leaves samples showed high antioxidant activity using DPPH as compared to non-transgenic control plants. With regard to RC50 values (the concentration of antioxidant required to achieve absorbance equal to 50% that of a control containing no antioxidants), RS3 transgenic lines showed highest radical-scavenging abilities (RC50 = 16.00 ± 2.00 µm). The DPPH free radical scavenging and LDL peroxidation activities of trans-3’-H-Rglu and trans-resveratrol isolated from transgenic R. glutinosa evaluated (Fig. 2& 3). DPPH activity of trans-resveratrol were significantly higher (72 ± 4.5 µm) than trans-3’-H-Rglu (198 ± 6.8 µm). This could be attributed to the higher level of accumulation of resveratrol compounds in the transgenic R. glutinosa (Fig. 4).
2.2.2. Superoxide Dismutase (SOD) activity of transgenic
The SOD activities non transgenic plant and transgenic plants (without water stress) were 13.81 and 11.23% respectively. In contrast, the SOD activities non transgenic plant and transgenic plants (with water stress) were 24.59 and 3.8 % respectively (Fig. 5).
2.2.3. Phenolic compound analysis of transgenic
The quantitative analysis of phenolic compounds of non-transgenic and transgenic
The average total concentrations of phenolic compounds in control plant stem and roots were 412.69 and 210.94 µg/g dry weight (DW), respectively; in comparison, transgenic stem and root samples had higher concentrations of 468.27–537.88 and 189.01–360.01µg/g DW, respectively. The phenolic compounds that increased in the transgenic lines were
2.2.4. Antimicrobial activities transgenic
Antimicrobial activities of the non-transgenic and transgenic plants were assessed by a paper disc diffusion assay. The results indicated variation in the antimicrobial properties of the resveratrol-3-O-B-D glucoside and resveratrol extracted from the transgenic
|Compounds||Clear zone (mm)|
|Conc. (ppm)||P. jadinii||C. albicans||S. aureus||B. subtilis||K.
2.2.5. Morphological characterization of transgenic
Phenotypic differences were observed within the different transgenic lines and between the transgenic and non-transgenic control plants (Table 4). However, there were no apparent differences in terms of root length and root diameter. Significant differences in root weight were observed between transgenic and non-transgenic lines and showed reduced weight over control plants.
|Line||Root length (cm)||root diameter||Root weight|
2.2.6. Analysis of catapol content of transgenic
The catapol contents and composition in subterranean parts of the transgenic lines and non-transformed plants were investigated using HPLC (Fig. 6). Overexpression of RS3 gene significantly increased the catapol, compared to that of wild type
2.2.7. Effect of the photosynthesis rate in transgenic
To compare the effect of RS3 gene overexpression on the photosynthesis rate and yield of transgenic and control plants, we measured stomatal conductance (gs), CO2 concentration (CI), and photosynthesis rate (A) and found significant differences in these factors between transgenic and control plants (Table 5). The photosynthesis rate increased progressively with increasing CO2 concentration. Photosynthesis rate of both non-transgenic and transgenic plants reduced by the increased duration of dry stress, being much lower at 15 days. Comparatively, transgenic lines showed higher photosynthetic control plants. Therefore, it is very possible that the higher level of RS3 gene in the transgenic plant is responsible for its enhanced photosynthetic performance.
|Non-transgenic plant||Transgenic plant|
|Control-||14.63 ± 0.1||0.29 ± 0.0||241.67
|17.42 ± 0.14||0.27 ± 0.02||212.47
|Dry stress||3D||9.68 ± 0.06||0.11 ± 0.01||195.8
|17.1 ± 0.06||0.32 ± 0.0||223.98
|9D||3.63 ± 0.14||0.02 ± 0.0||45.52
|14.39 ± 0.04||0.23 ± 0.0||220.13
|15D||1.13 ± 0.01||0.0||215.82
|1.69 ± 0.31||0.0||194.13
It can be concluded that introduction of RS3 gene into the
Anwart WL, Porter PM, Granato TC, Hassett JJ 1985HPLC separation and wavelength area ratios of more than 50 phenolics and flavonoids. J. Chem. Ecol. 11 383 395.
Awika J. M. Rooney L. W. 2004Sorghum phytochemical and their potential impact on human health. Phytochemistry, 65 1199 1221.
Beyer Jr W. E. Fridovich I. 1987 Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions.Anal. Biochem. 161 559 566.
Buege JA, Aust SD 1978 Microsomal lipid peroxidation.Methods Enzymol. 52 302 310.
Cal C. Garban H. Jazirehi A. Yeh C. Mizutani Y. Bonavida B. 2003 Resveratrol and cancer: chemoprevention, apoptosis, and chemo-immunosensitizing activities.Curr. Med. Chem. Anti-Canc. Agents. 3 77 93.
Gonsalves 1998 Control of papaya ringspot virus in papaya: A case study., 36 415 437.
Gordana S. C. Jasna M. C. MD Sonja Tumbas. V. T. Markov S. L. Dragoljub D. C. 2007Antioxidant potential, lipid peroxidation inhibition and antimicrobial activities of Satureja montana L. Subsp. kitaibelii extracts. International Journal of Molecular Sciences, 8 1013 1027.
Hain R. Reif H. J. Krause E. Langebartels R. Kind H. Vornam B. Wiese W. Schmelzer E. Schreier P. H. Stocker R. H. Stenzel K. 1993Disease resistance results from phytoalexin expression in a novel plant. Nature 361 153 156.
Ivan Y. Tsonko T. Violeta V. Katya G. Peter I. Nikolai T. Tatyana P. 2001Changes in CO2 ssimilation, transpiration and stomatal resistance of different wheat cultivars experiencing drought under field conditions. Bulg J Plant Physiol 27:20-33
Ignatowicz E. Baer-Dubowska W. 2001Resveratrol, a natural chemopreventive agent against degenerative diseases. Pol. J. Pharmacol. 53 557 569.
Jang M. Cai L. Udean G. O. Slowing K. V. Thomas C. F. Beecher C. W. W. Fong H. H. S. Farnsworth N. R. Kinghorn A. D. Mehta R. G. Moon R. C. Pezzuto J. M. 1997Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275 218 220.
Lim J. D. Yun S. J. Lee S. J. Chung I. M. MJ Kim Heo. K. Yu C. Y. 2004Comparison of resveratrol contents in medicinal plants. Korean J. Medicinal Crop Sci. 12 163 170.
Pinto Y. M. Kok R. A. Baulcombe D. C. 1999Resistance to rice yellow mottle virus (RYMV) in cultivated African rice varieties containing RYMV transgenes. Nature Biotechnology 17 702 707.
Thanavala Y. Yang Y. Lyons P. Mason H. S. Arntzen C. 1995Immunogenicity of Transgenic Plant-derived Hepatitis B Surface Antigen. Proc. Nat. Acad. Sci. USA. 92 3358 3361.
Thomzik J. E. Stenzel K. Stocker R. Schreier P. H. Hain R. Stahl D. J. 1997Synthesis of a grapevine phytoalexin in transgenic tomatoes (Lycopersicon esculentum Mill.) conditions resistance against Phytophthora infestations. Physiol. Mol. Plant Pathol. 51 265 278.
WHO.: 1996aMicronutrient Malnutrition: Half theWorld’s Population Affected,World Health Organization, 13 Nov. 1996 78 1 4.
Q. Kadota S. Tadata T. Namba T. 1996Antioxidant effect of phenylethanoids from Biological and Pharmaceutical Bulletin. 19 1580 1585.
Ye X. et al. 2000Engineering the provitamin A (beta carotene) biosynthesis pathway into (carotenoid-free) rice endosperm. Science, 287 303 305.
Zhuang H, Kim YS, Koehler RC and Dore S( 2003) Potential mechanism by which resveratrol, a red wine constituent, protects neurons. Ann. New York Acad. Sci. 993 276 286.