Funct Integr Genomics (2014) 14:237–244 DOI 10.1007/s10142-013-0355-y
ORIGINAL PAPER
The substantive equivalence of transgenic (Bt and Chi ) and non-transgenic cotton based on metabolite profiles Bentol Hoda Modirroosta & Masoud Tohidfar & Jalal Saba & Foad Moradi
Received: 3 April 2013 / Revised: 2 December 2013 / Accepted: 5 December 2013 / Published online: 28 December 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Compositional studies comparing transgenic with non-transgenic counterpart plants are almost universally required by governmental regulatory bodies. In the present study, two T2 transgenic cotton lines containing chitinase (Line 11/57) and Bt lines (Line 61) were compared with non-transgenic counterpart. To do this, biochemical characteristics of leaves and seeds, including amino acids, fatty acids, carbohydrates, anions, and cations contents of the studied lines were analyzed using GC/MS, high-performance liquid chromatography (HPLC), and ion chromatography (IC) analyzers, respectively. polymerase chain reaction (PCR) and Western blot analyses confirmed the presence and expression of Chi and Bt genes in the studied transgenic lines. Although, compositional analysis of leaves contents confirmed no significant differences between transgenic and non-transgenic counterpart lines, but it was shown that glucose content of chitinase lines, fructose content of transgenic lines (Bt and chitinase) and asparagine and glutamine of chitinase lines were significantly higher than the non-transgenic counterpart plants. Both the transgenic lines (Bt and chitinase) showed significant decrease in the amounts of sodium in comparison to the non-transgenic counterpart plants. The experiments on the seeds showed that histidine, isoleucine, leucine, and phenylalanine contents of all transgenic and non-transgenic lines were the same, whereas other amino acids were significantly increased in the transgenic lines. Surprisingly, it was observed that the concentrations of stearic acid, myristic acid, oleic acid, and linoleic acid in the chitinase line were significantly B. H. Modirroosta : J. Saba Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Zanjan, Zanjan, Islamic Republic of Iran M. Tohidfar (*) : F. Moradi Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj 3135923151, Islamic Republic of Iran e-mail: [email protected]
different than those of non-transgenic counterpart plants, but these components were the same in both Bt line and its nontransgenic counterpart. It seems that more changes observed in the seed contents than leaves is via this point that seeds are known as metabolites storage organs, so they show greater changes in the metabolites contents comparing to the leaves. Keywords Transgenic cotton . Metabolite . HPLC . GC/MS . IC
Introduction Genetic engineering has been employed for several years to enhance crop yields and minor pesticide use. Genetically modified (GM) plants with improved resistance to insect pests have been commercialized for over a decade and are widely used universal (Raybould et al. 2010). Transgenic cotton (Gossypium hirsutum) with resistance to Lepidopteran insects and tolerance to fungal diseases has been developed in the laboratory (Tohidfar et al. 2008, 2012a). Genetically engineered plants almost sustain undesirable genetic changes other than the transformation goals. Differences between transgenic and non-transgenic plants may result from the insertion of the exogenous genes or from the subsequent tissue culture, in which case they are called insertion mutations and somaclonal variations (Larkin and Scowcroft, 1981). Molecular large-scale profiling technologies, i.e., transcriptomics, proteomics, and metabolomics are useful tools for evaluating compositional changes in transgenic crops due to gene transformation (Baudo et al. 2006). A targeted analysis of the key nutrients and anti-nutrients has been used to assess the potentially unintended effects caused by the genetic modification (Shepherd et al. 2006). Compositional studies over a decade highlight the compositional similarities of transgenic crops with their non-transgenic
238
counterparts, and it was used for biosafety studies of different transgenic plants, such as rice, soybean, cotton, corn, alfalfa, and wheat (Larkin and Harrigan 2007). The codex alimentarius elaborated principles of “substantial equivalence” to detect the possible unexpected effects of GM crops, which is a crucial step in the safety evaluation and toxicological assessment (Codex, 2003). A powerful reason indicating safety of the GM crops is the lack of statistically significant difference in the concentration of the key metabolites in the transgenic crop comparing to its non-transgenic counterpart. However, detection of statistically considerable variation does not necessarily indicate that GMO is not safe (Raybould et al. 2010). The dissimilarity between the concentration of transgenic and non-transgenic counterpart lines can be considered biologically insignificant, if the concentration in the transgenic crop is within the range for the crop generally albeit the difference is statistically significant (Raybould et al. 2010). In the previous, our studies (Tohidfar et al. 2008; Tohidfar et al. 2012a;), two genes cry1Ab and Chi were transferred into cotton using Agrobacterium tumefaciens. The achieved lines showed resistance to cotton bollworm (Helicoverpa armigera) and verticillium wilt disease (Verticillium dahliae) at laboratory and greenhouse bioassays, and gene stability and expression were observed up to T2 generation. The objective of the present study was to evaluate substantial equivalence of the metabolites (amino acid, fatty acid, carbohydrate, cation, and anion) in the transgenic and their non-transgenic counterpart plants, and the stability of the gene integration and expression using molecular studies.
Materials and methods Plant material Two transgenic T2 cotton lines including Bt (Line 61) and chitinase (line 57) lines (Tohidfar et al. 2008, 2012a) and nontransgenic counterpart were used in the study. Germinated seeds were placed in pots under controlled temperature and watering conditions and natural light in the greenhouse. Leaf samples of three replications were taken from 5-weeks-old plants for metabolic analysis. Cotton seed samples were harvested, ginned, prepared, and analyzed for composition. DNA extraction and polymerase chain reaction analysis Total DNA was isolated from fresh leaves as previously described by Li et al. (2001). Polymerase chain reaction (PCR) was performed using specific primers for both genes (Tohidfar et al., 2008, 2012a), and according to the following steps: 5 min at 95 °C, 30 cycles; 30 s at 94 °C, 1 min at 60 °C, and 1 min at 72 °C for Chi gene; 4 min at 94 °C, 35 cycles; and
Funct Integr Genomics (2014) 14:237–244
1 min at 94 °C, 1 min at 60 °C, and 3 min at 72 °C for cry1Ab gene, followed by a final extension at 72 °C for 5 min. Expected PCR products size were about 870 bp for Chi gene and 785 bp for cry1Ab gene. Protein extraction and Western blot analysis Protein was extracted from fresh leave samples (ground in liquid nitrogen with a mortar and pestle) with 1 ml of extraction buffer [40 % (w/v) SDS, 5 % (v/v) 2-mercaptoethanol, 20 % (v/v) glycerol, 68 mM Tris-HCl (pH 6.8)] according to Tohidfar et al. (2012a)). Protein extracts were clarified at 10,000 rpm for 10 min at 4 °C. Supernatants were saved and protein concentration was determined by the Bradford method (Bradford, 1976). The extracts (50 μg of protein) were separated by SDS-PAGE electrophoresis and subjected to immunoblot analysis. The separated proteins, were electrophoretically transferred onto a Hybond-C membrane (Amersham, Buckinghamshire, England) and exposed to the anti-Cry1Ab for Bt line (kindly provided by Pro. Altosar, University of Ottawa, Canada) and anti-serum and the rabbit anti-chi-I antiserum (kindly provided by Dr. Els van Deventer, Zeneca Mogen International, Leiden, The Netherlands) for chitinase line (at a dilution of 1:2000 v/v). Alkaline phosphataseconjugated goat anti-rabbit IgG was used as the secondary antibody. 5-Bromo-4- chloro-3-indolyl Phosphate (BCIP)/nitro blue tetrazolium (NBT) was used for visualization. Fatty acid analysis To extract the fatty acids from the grounded cotton seeds, the cotton seed powder was mixed with petroleum ether. After evaporating petroleum ether, the oil was kept at 4 °C for analysis. The extracted oil was combined with KOH to derivatization just before use. The fatty acid composition and concentration were analyzed by gas chromatography using CP-sil88 column, according to Rui et al. (2007). Helium was used as the carrier gas. The analysis was performed under the following temperature program: 1 min at 170 °C, followed by a 5 °C min−1 oven temperature ramp to 190 °C, and stayed for 30 min at this temperature, and finally by a 5 °C min−1 ramp to 210 °C and stayed for 22 min at this temperature. Carbohydrate analysis Carbohydrate was extracted from the dried leaves and dried cotton seeds using the ethanol. Briefly, dried material (0.2 g) was homogenized in 1.5 ml of 80 % ethanol. The supernatant was collected after centrifuging at 3,000 rpm for 5 min. The removed supernatant placed in oven to evaporate the ethanol. After evaporation, the following chemicals were added: 10 ml distilled water, 470 μl 0.3 N barium hydroxide, and500 μl 5 % zinc sulfate. After centrifuging the mixture at 3,000 rpm for
Funct Integr Genomics (2014) 14:237–244
10 min, the supernatant placed in oven to evaporate the aqueous phase. Then, samples were shaked for 30 s with 1 ml of distilled water and then filtered. The carbohydrates in leaves and cotton seeds were examined by highperformance liquid chromatography (HPLC; Hendrix and Peelen 1987). Amino acid analysis Leaf tissues from 7-week-old plants were harvested and flash frozen in the liquid N2. Also, the cotton seeds were used for extraction of the amino acids. The materials were grounded into fine powder using mortar and pestle. The powder (100 mg) was weighed and then, soaked in 1 ml of 80 % ethanol. After vortexing at 80 °C for 60 min, the samples were cooled to the room temperature and were centrifuged at 13,000 rpm for 10 min. The supernatant was concentrated to dryness in a concentrator system. The dried residue was resolved in 1 ml of distilled water and stored at −20 °C until amino acid analysis. Amino acids were derived from 250 μl of extracts by vigorous shaking for 120 s with borate buffer (2.85 g tetra borate in 100 ml distilled water) and OPA buffer (0.05 g Phthaldialdehyde in 4.5 ml methanol and 0.5 ml borate buffer and 50 μl 2-Mercaptoethanol). The extracts were determined after mixing with 0.5 M HCl (50 μl) via HPLC. Separation of amino acids on HPLC with HALO C18 column was accomplished with fluorescence detector (Marur et al. 1994). Cation and anion analysis Soluble cations were extracted from dried leaves (0.2 g) by shaking for 4 h with 25 ml extraction solvent (10 % acetic acid and 0.1 N nitric acid) at 80 °C water bath. After cooling to room temperature for 24 h, the samples were then filtered and subjected to ion chromatography (IC; 850 Professional IC, Methrohm, Switzerland) with a Metrosep C2 250 column. Dried leaf tissues were ground into powder (0.2 g) and were extracted with distilled water (20 ml) for 24 h at 80 °C water bath to determine the content of anions. The extract was filtered and subjected to the same IC but with a Metrosep Asupp7 column (Metrohm company 2011).
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transgenic counterpart plants were negative for the Chi and cry1Ab genes. These results indicated that both Chi and cry1Ab genes were inherited to the studied transgenic plants. Detection of Cry1Ab protein in the T2 generation of Bt line was verified by the appearance of a 67-KDa band in immunoblot analysis (Fig. 2a). Also, the chitinase protein activity in the transgenic plants was assayed and revealed by Western blotting. The results revealed that a band of expected 32 kDa size in the chitinase transgenic plants (Fig. 2b). Expression of the Chi and cry1Ab genes in the transgenic plants indicates the stable expression of the transgenes. Non-transgenic counterpart plants did not display any signal for the Cry1Ab and chitinase proteins. Based on the previous studies, when the chitinase line (11/ 57) was digested with XbaI, two bands were detected for the transgenic line. Since there was only one XbaI site in the TDNA (Fig. 3a), this finding revealed the presence of two copies of the transgene in line 57 (Tohidfar et al. 2012b). Also, digested DNA extracted from Bt line (61) with EcoRI was showed only one band. Since there is only one EcoRI site in the T-DNA (Fig. 3b), this result indicates the presence of only one single copy of the transgene in the Bt line (61) (Tohidfar et al. 2008). Fatty acids analysis Fatty acid profiles were evaluated in the transgenic and nontransgenic cotton seeds (Table 1). It was shown that some fatty acids contents in the chitinase and Bt lines were significantly different from their non-transgenic counterpart (P≤0.05). For instance, stearic acid, myristic acid, oleic acid, and linoleic acid concentrations were 0.15, 0.56, 20.8, and 56.4 % in the chitinase line, whereas 0.33, 0.36, 18.2, and 59.9 % in the non-transgenic plants, respectively. It is important to note that all the differences observed in the study (exception for stearic acid contents) were within the International Life Sciences Institute (Life Sciences Institute) 2006) reported ranges for cotton seed (crop composition database, available at http:// www.cropcomposition.org); therefore, they were regarded as biologically non-significant. These data showed that there were no significant difference between the Bt cotton line and its non-transgenic counterpart (Table 1).
Results
Carbohydrate analysis
Molecular analyses
There was no significant difference between the sucrose and glucose contents of the Bt and chitinase transgenic and nontransgenic counterpart cotton leaf extracts (Table 2). Surprisingly, the glucose content of the Bt lines and non-transgenic counterpart were the same and no significant differences was observed; whereas, glucose content of the chitinase plants and the non-transgenic counterpart plants were significantly
To evaluate transformation at the molecular level, the genomic DNA of transgenic plants were screened by PCR with specific primers. Amplification of 785 and 870 bp fragments produced from DNA samples of transgenic Bt and chitinase lines, respectively (Fig. 1). Non-
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Funct Integr Genomics (2014) 14:237–244
Fig. 1 PCR analysis of the transgenic cotton plants using specific primer pairs in agarose gel: a Bt plants. Lanes 1–11, DNA from transgenic cotton lines; lane 12, DNA from non-transgenic counterpart; lane 13, DNA from
pBI121-Cry1Ab; M, 1.0 kb DNA ladder. b chitinase plants. Lanes 1–9, DNA from transgenic cotton lines; lane 10, DNA from non-transgenic counterpart; lane 11, DNA from pBI121-BCH; M, 1.0 kb DNA ladder
different (Table 2). In addition, the results showed that the leaves fructose content of both the transgenic lines was significantly higher than the non-transgenic counterpart (Table 2). It was confirmed that the carbohydrates contents (sucrose and glucose) of the chitinase and non-transgenic lines seeds were significantly different. Also glucose, fructose, and sucrose contents of the Bt cotton and the non-transgenic counterpart seeds were significantly different (Table 3).
different from those of the non-transgenic counterpart, but contents of four amino acids, including histidine, phenylalanine, isoleucine, and leucine were the same in both transgenic and the non-transgenic counterpart seeds. Interestingly, the contents of all 20 amino acids of Bt cotton line seeds were significantly increased comparing to the non-transgenic counterpart seeds (Table 5).
Cations analysis Amino acids analysis Out of 20 measured amino acids, 18 amino acids had no significant differences as compared to the non-transgenic counterpart leaf extracts (Table 4), but, asparagine and glutamine contents were significantly changed in the chitinase plants. However amino acids of Bt line had no significant difference compared to the non-transgenic counterpart. Experiments on the seeds showed that 16 amino acids contents of the chitinase plants seeds were significantly
Fig. 2 Western immunoblot analysis of the transgenic cotton lines leaf extracts. a Bt plants: lanes 1–6, protein extracts from transgenic Bt cotton lines; lane 7, protein extract from non-transgenic counterpart cotton; M, prestained standard markers in dalton; and lane 8, Cry1Ab protein
The simultaneous assay of the mono and divalent cations was performed using IC. The quantitative values of the identified cations in the cotton studied leaves are presented in Table 6. Although the transgenic lines exhibited significant decreased in the amounts of sodium, but no significant differences were detected for the other cations. Results revealed that Bt and chitinase transgenic lines did not show any significant different for the amount of K+, Mg2+, and NH4+cationsas comparing to the non-transgenic counterpart line.
standard. b Chitinase plants: lanes 1–3 and 5–8, protein extracts from transgenic chitinase cotton lines; lane 4, protein extract from non-transgenic counterpart cotton; and M, prestained standard markers in kDa
Funct Integr Genomics (2014) 14:237–244
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Fig. 3 Chimeric gene map of the recombinant binary vector: a pBI121BCH carrying the bean Chi gene and nptII gene driven by CaMV35S promoter (P35S). LB left border, RB right border, nptII neomycin phosphotransferase, Chi chitinase, Pnos nopaline synthase promoter, Tnos
nopaline synthase terminator. b pBI121-Cry1Ab carrying the cry1Ab and nptII genes driven by the CaMV35S promoter. LB left border, RB right border, nptII neomycin phosphotransferase, Nos-ter nopaline synthase terminator
Anions analysis
the cotton seeds would represent a potential health benefit and may expand utilization of raw cotton seed (Chapman et al., 2001). To generally reflect the metabolic variations related to transformation and breeding, metabolite profiling might be necessary. According to the results of the present study, the quantity of some metabolites were identified significantly different between transgenic and non-transgenic counterpart, which might be as result of position side effects of the transgene which needs more investigations. Little variations were detected in the fatty acid contents of chitinase line, but these values were within the Crop Composition Database ranges available for cotton seed oil (ILSI Life Sciences Institute) 2006). Hence, the insertion of Bt (Line 61) and chitinase (Line 57) genes to cotton had no effect on the oil quantity and quality of the transgenic lines compared to the non-transgenic counterpart (Table 1). The results reported by Berberich et al. (1996), Nida et al. (1996), and Bertrand et al.(2005) confirmed that the highest quantity of the fatty acids belongs to linoleic acid, palmitic acid, and oleic acid, respectively, which is in accordance to our results. Berberich et al. (1996) did not observe any differences in fatty acid contents of the studied transgenic and non-transgenic cotton seeds, but the results of Nida et al. (1996) showed minor differences for oleic and palmitic acids.
All the major nutrient anions could be easily quantified in the water-extracted samples, with superior sensitivity and reproducibility. Average values determined for aqueous extracts of the studied samples are given in Table 7. The results revealed that there were no statistically significant differences in the levels of the measured anions of transgenic lines comparing to the non-transgenic counterpart. All the anions, including acetate, chloride, nitrate, phosphate, sulfate, succinate, and oxalate in both Bt and chitinase transgenic lines had no considerable variation comparing to the non-transgenic counterpart.
Discussion Transgenes and somaclonal variations related to the regeneration process could be causes of metabolic variations between the transgenic and non-transgenic counterpart (Zhou et al. 2012). Also, the metabolic variations between the transgenic plant and its non-transgenic counterpart might be due to the position side effects of gene inserted; however, these unintended variations might be good. For instance, increased oleic acid content with a compensatory reduction in linoleic acid in
Table 1 Fatty acid composition (%) in cotton seed of Bt, chitinase, and non-transgenic counterpart lines Non-transgenic counterpart
chitinase line
Bt line
ILSI
Myristic acid Palmitic acid Stearic acid Oleic acid
0.3614±0.07b 21.19±0.38a 0.3310±0.18a 18.20±0.85b
0.5612±0.07a 22.08±1.12a 0.1545±0.05b 20.78±0.95a
0.4089±0.05b 21.21±1.09a 0.2354±0.1ab 18.73±1.18b
0.45–2.4* 15.11–27.9 0.2–3.11 12.8–25.3
Linoleic acid
59.91±1.37a
56.38±1.17b
59.41±1.59a
46.0–59.4
Values are presented as mean±SD SD standard deviation Superscript letters indicate fatty acids that show statistically significant differences (P
ORIGINAL PAPER
The substantive equivalence of transgenic (Bt and Chi ) and non-transgenic cotton based on metabolite profiles Bentol Hoda Modirroosta & Masoud Tohidfar & Jalal Saba & Foad Moradi
Received: 3 April 2013 / Revised: 2 December 2013 / Accepted: 5 December 2013 / Published online: 28 December 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Compositional studies comparing transgenic with non-transgenic counterpart plants are almost universally required by governmental regulatory bodies. In the present study, two T2 transgenic cotton lines containing chitinase (Line 11/57) and Bt lines (Line 61) were compared with non-transgenic counterpart. To do this, biochemical characteristics of leaves and seeds, including amino acids, fatty acids, carbohydrates, anions, and cations contents of the studied lines were analyzed using GC/MS, high-performance liquid chromatography (HPLC), and ion chromatography (IC) analyzers, respectively. polymerase chain reaction (PCR) and Western blot analyses confirmed the presence and expression of Chi and Bt genes in the studied transgenic lines. Although, compositional analysis of leaves contents confirmed no significant differences between transgenic and non-transgenic counterpart lines, but it was shown that glucose content of chitinase lines, fructose content of transgenic lines (Bt and chitinase) and asparagine and glutamine of chitinase lines were significantly higher than the non-transgenic counterpart plants. Both the transgenic lines (Bt and chitinase) showed significant decrease in the amounts of sodium in comparison to the non-transgenic counterpart plants. The experiments on the seeds showed that histidine, isoleucine, leucine, and phenylalanine contents of all transgenic and non-transgenic lines were the same, whereas other amino acids were significantly increased in the transgenic lines. Surprisingly, it was observed that the concentrations of stearic acid, myristic acid, oleic acid, and linoleic acid in the chitinase line were significantly B. H. Modirroosta : J. Saba Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Zanjan, Zanjan, Islamic Republic of Iran M. Tohidfar (*) : F. Moradi Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj 3135923151, Islamic Republic of Iran e-mail: [email protected]
different than those of non-transgenic counterpart plants, but these components were the same in both Bt line and its nontransgenic counterpart. It seems that more changes observed in the seed contents than leaves is via this point that seeds are known as metabolites storage organs, so they show greater changes in the metabolites contents comparing to the leaves. Keywords Transgenic cotton . Metabolite . HPLC . GC/MS . IC
Introduction Genetic engineering has been employed for several years to enhance crop yields and minor pesticide use. Genetically modified (GM) plants with improved resistance to insect pests have been commercialized for over a decade and are widely used universal (Raybould et al. 2010). Transgenic cotton (Gossypium hirsutum) with resistance to Lepidopteran insects and tolerance to fungal diseases has been developed in the laboratory (Tohidfar et al. 2008, 2012a). Genetically engineered plants almost sustain undesirable genetic changes other than the transformation goals. Differences between transgenic and non-transgenic plants may result from the insertion of the exogenous genes or from the subsequent tissue culture, in which case they are called insertion mutations and somaclonal variations (Larkin and Scowcroft, 1981). Molecular large-scale profiling technologies, i.e., transcriptomics, proteomics, and metabolomics are useful tools for evaluating compositional changes in transgenic crops due to gene transformation (Baudo et al. 2006). A targeted analysis of the key nutrients and anti-nutrients has been used to assess the potentially unintended effects caused by the genetic modification (Shepherd et al. 2006). Compositional studies over a decade highlight the compositional similarities of transgenic crops with their non-transgenic
238
counterparts, and it was used for biosafety studies of different transgenic plants, such as rice, soybean, cotton, corn, alfalfa, and wheat (Larkin and Harrigan 2007). The codex alimentarius elaborated principles of “substantial equivalence” to detect the possible unexpected effects of GM crops, which is a crucial step in the safety evaluation and toxicological assessment (Codex, 2003). A powerful reason indicating safety of the GM crops is the lack of statistically significant difference in the concentration of the key metabolites in the transgenic crop comparing to its non-transgenic counterpart. However, detection of statistically considerable variation does not necessarily indicate that GMO is not safe (Raybould et al. 2010). The dissimilarity between the concentration of transgenic and non-transgenic counterpart lines can be considered biologically insignificant, if the concentration in the transgenic crop is within the range for the crop generally albeit the difference is statistically significant (Raybould et al. 2010). In the previous, our studies (Tohidfar et al. 2008; Tohidfar et al. 2012a;), two genes cry1Ab and Chi were transferred into cotton using Agrobacterium tumefaciens. The achieved lines showed resistance to cotton bollworm (Helicoverpa armigera) and verticillium wilt disease (Verticillium dahliae) at laboratory and greenhouse bioassays, and gene stability and expression were observed up to T2 generation. The objective of the present study was to evaluate substantial equivalence of the metabolites (amino acid, fatty acid, carbohydrate, cation, and anion) in the transgenic and their non-transgenic counterpart plants, and the stability of the gene integration and expression using molecular studies.
Materials and methods Plant material Two transgenic T2 cotton lines including Bt (Line 61) and chitinase (line 57) lines (Tohidfar et al. 2008, 2012a) and nontransgenic counterpart were used in the study. Germinated seeds were placed in pots under controlled temperature and watering conditions and natural light in the greenhouse. Leaf samples of three replications were taken from 5-weeks-old plants for metabolic analysis. Cotton seed samples were harvested, ginned, prepared, and analyzed for composition. DNA extraction and polymerase chain reaction analysis Total DNA was isolated from fresh leaves as previously described by Li et al. (2001). Polymerase chain reaction (PCR) was performed using specific primers for both genes (Tohidfar et al., 2008, 2012a), and according to the following steps: 5 min at 95 °C, 30 cycles; 30 s at 94 °C, 1 min at 60 °C, and 1 min at 72 °C for Chi gene; 4 min at 94 °C, 35 cycles; and
Funct Integr Genomics (2014) 14:237–244
1 min at 94 °C, 1 min at 60 °C, and 3 min at 72 °C for cry1Ab gene, followed by a final extension at 72 °C for 5 min. Expected PCR products size were about 870 bp for Chi gene and 785 bp for cry1Ab gene. Protein extraction and Western blot analysis Protein was extracted from fresh leave samples (ground in liquid nitrogen with a mortar and pestle) with 1 ml of extraction buffer [40 % (w/v) SDS, 5 % (v/v) 2-mercaptoethanol, 20 % (v/v) glycerol, 68 mM Tris-HCl (pH 6.8)] according to Tohidfar et al. (2012a)). Protein extracts were clarified at 10,000 rpm for 10 min at 4 °C. Supernatants were saved and protein concentration was determined by the Bradford method (Bradford, 1976). The extracts (50 μg of protein) were separated by SDS-PAGE electrophoresis and subjected to immunoblot analysis. The separated proteins, were electrophoretically transferred onto a Hybond-C membrane (Amersham, Buckinghamshire, England) and exposed to the anti-Cry1Ab for Bt line (kindly provided by Pro. Altosar, University of Ottawa, Canada) and anti-serum and the rabbit anti-chi-I antiserum (kindly provided by Dr. Els van Deventer, Zeneca Mogen International, Leiden, The Netherlands) for chitinase line (at a dilution of 1:2000 v/v). Alkaline phosphataseconjugated goat anti-rabbit IgG was used as the secondary antibody. 5-Bromo-4- chloro-3-indolyl Phosphate (BCIP)/nitro blue tetrazolium (NBT) was used for visualization. Fatty acid analysis To extract the fatty acids from the grounded cotton seeds, the cotton seed powder was mixed with petroleum ether. After evaporating petroleum ether, the oil was kept at 4 °C for analysis. The extracted oil was combined with KOH to derivatization just before use. The fatty acid composition and concentration were analyzed by gas chromatography using CP-sil88 column, according to Rui et al. (2007). Helium was used as the carrier gas. The analysis was performed under the following temperature program: 1 min at 170 °C, followed by a 5 °C min−1 oven temperature ramp to 190 °C, and stayed for 30 min at this temperature, and finally by a 5 °C min−1 ramp to 210 °C and stayed for 22 min at this temperature. Carbohydrate analysis Carbohydrate was extracted from the dried leaves and dried cotton seeds using the ethanol. Briefly, dried material (0.2 g) was homogenized in 1.5 ml of 80 % ethanol. The supernatant was collected after centrifuging at 3,000 rpm for 5 min. The removed supernatant placed in oven to evaporate the ethanol. After evaporation, the following chemicals were added: 10 ml distilled water, 470 μl 0.3 N barium hydroxide, and500 μl 5 % zinc sulfate. After centrifuging the mixture at 3,000 rpm for
Funct Integr Genomics (2014) 14:237–244
10 min, the supernatant placed in oven to evaporate the aqueous phase. Then, samples were shaked for 30 s with 1 ml of distilled water and then filtered. The carbohydrates in leaves and cotton seeds were examined by highperformance liquid chromatography (HPLC; Hendrix and Peelen 1987). Amino acid analysis Leaf tissues from 7-week-old plants were harvested and flash frozen in the liquid N2. Also, the cotton seeds were used for extraction of the amino acids. The materials were grounded into fine powder using mortar and pestle. The powder (100 mg) was weighed and then, soaked in 1 ml of 80 % ethanol. After vortexing at 80 °C for 60 min, the samples were cooled to the room temperature and were centrifuged at 13,000 rpm for 10 min. The supernatant was concentrated to dryness in a concentrator system. The dried residue was resolved in 1 ml of distilled water and stored at −20 °C until amino acid analysis. Amino acids were derived from 250 μl of extracts by vigorous shaking for 120 s with borate buffer (2.85 g tetra borate in 100 ml distilled water) and OPA buffer (0.05 g Phthaldialdehyde in 4.5 ml methanol and 0.5 ml borate buffer and 50 μl 2-Mercaptoethanol). The extracts were determined after mixing with 0.5 M HCl (50 μl) via HPLC. Separation of amino acids on HPLC with HALO C18 column was accomplished with fluorescence detector (Marur et al. 1994). Cation and anion analysis Soluble cations were extracted from dried leaves (0.2 g) by shaking for 4 h with 25 ml extraction solvent (10 % acetic acid and 0.1 N nitric acid) at 80 °C water bath. After cooling to room temperature for 24 h, the samples were then filtered and subjected to ion chromatography (IC; 850 Professional IC, Methrohm, Switzerland) with a Metrosep C2 250 column. Dried leaf tissues were ground into powder (0.2 g) and were extracted with distilled water (20 ml) for 24 h at 80 °C water bath to determine the content of anions. The extract was filtered and subjected to the same IC but with a Metrosep Asupp7 column (Metrohm company 2011).
239
transgenic counterpart plants were negative for the Chi and cry1Ab genes. These results indicated that both Chi and cry1Ab genes were inherited to the studied transgenic plants. Detection of Cry1Ab protein in the T2 generation of Bt line was verified by the appearance of a 67-KDa band in immunoblot analysis (Fig. 2a). Also, the chitinase protein activity in the transgenic plants was assayed and revealed by Western blotting. The results revealed that a band of expected 32 kDa size in the chitinase transgenic plants (Fig. 2b). Expression of the Chi and cry1Ab genes in the transgenic plants indicates the stable expression of the transgenes. Non-transgenic counterpart plants did not display any signal for the Cry1Ab and chitinase proteins. Based on the previous studies, when the chitinase line (11/ 57) was digested with XbaI, two bands were detected for the transgenic line. Since there was only one XbaI site in the TDNA (Fig. 3a), this finding revealed the presence of two copies of the transgene in line 57 (Tohidfar et al. 2012b). Also, digested DNA extracted from Bt line (61) with EcoRI was showed only one band. Since there is only one EcoRI site in the T-DNA (Fig. 3b), this result indicates the presence of only one single copy of the transgene in the Bt line (61) (Tohidfar et al. 2008). Fatty acids analysis Fatty acid profiles were evaluated in the transgenic and nontransgenic cotton seeds (Table 1). It was shown that some fatty acids contents in the chitinase and Bt lines were significantly different from their non-transgenic counterpart (P≤0.05). For instance, stearic acid, myristic acid, oleic acid, and linoleic acid concentrations were 0.15, 0.56, 20.8, and 56.4 % in the chitinase line, whereas 0.33, 0.36, 18.2, and 59.9 % in the non-transgenic plants, respectively. It is important to note that all the differences observed in the study (exception for stearic acid contents) were within the International Life Sciences Institute (Life Sciences Institute) 2006) reported ranges for cotton seed (crop composition database, available at http:// www.cropcomposition.org); therefore, they were regarded as biologically non-significant. These data showed that there were no significant difference between the Bt cotton line and its non-transgenic counterpart (Table 1).
Results
Carbohydrate analysis
Molecular analyses
There was no significant difference between the sucrose and glucose contents of the Bt and chitinase transgenic and nontransgenic counterpart cotton leaf extracts (Table 2). Surprisingly, the glucose content of the Bt lines and non-transgenic counterpart were the same and no significant differences was observed; whereas, glucose content of the chitinase plants and the non-transgenic counterpart plants were significantly
To evaluate transformation at the molecular level, the genomic DNA of transgenic plants were screened by PCR with specific primers. Amplification of 785 and 870 bp fragments produced from DNA samples of transgenic Bt and chitinase lines, respectively (Fig. 1). Non-
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Fig. 1 PCR analysis of the transgenic cotton plants using specific primer pairs in agarose gel: a Bt plants. Lanes 1–11, DNA from transgenic cotton lines; lane 12, DNA from non-transgenic counterpart; lane 13, DNA from
pBI121-Cry1Ab; M, 1.0 kb DNA ladder. b chitinase plants. Lanes 1–9, DNA from transgenic cotton lines; lane 10, DNA from non-transgenic counterpart; lane 11, DNA from pBI121-BCH; M, 1.0 kb DNA ladder
different (Table 2). In addition, the results showed that the leaves fructose content of both the transgenic lines was significantly higher than the non-transgenic counterpart (Table 2). It was confirmed that the carbohydrates contents (sucrose and glucose) of the chitinase and non-transgenic lines seeds were significantly different. Also glucose, fructose, and sucrose contents of the Bt cotton and the non-transgenic counterpart seeds were significantly different (Table 3).
different from those of the non-transgenic counterpart, but contents of four amino acids, including histidine, phenylalanine, isoleucine, and leucine were the same in both transgenic and the non-transgenic counterpart seeds. Interestingly, the contents of all 20 amino acids of Bt cotton line seeds were significantly increased comparing to the non-transgenic counterpart seeds (Table 5).
Cations analysis Amino acids analysis Out of 20 measured amino acids, 18 amino acids had no significant differences as compared to the non-transgenic counterpart leaf extracts (Table 4), but, asparagine and glutamine contents were significantly changed in the chitinase plants. However amino acids of Bt line had no significant difference compared to the non-transgenic counterpart. Experiments on the seeds showed that 16 amino acids contents of the chitinase plants seeds were significantly
Fig. 2 Western immunoblot analysis of the transgenic cotton lines leaf extracts. a Bt plants: lanes 1–6, protein extracts from transgenic Bt cotton lines; lane 7, protein extract from non-transgenic counterpart cotton; M, prestained standard markers in dalton; and lane 8, Cry1Ab protein
The simultaneous assay of the mono and divalent cations was performed using IC. The quantitative values of the identified cations in the cotton studied leaves are presented in Table 6. Although the transgenic lines exhibited significant decreased in the amounts of sodium, but no significant differences were detected for the other cations. Results revealed that Bt and chitinase transgenic lines did not show any significant different for the amount of K+, Mg2+, and NH4+cationsas comparing to the non-transgenic counterpart line.
standard. b Chitinase plants: lanes 1–3 and 5–8, protein extracts from transgenic chitinase cotton lines; lane 4, protein extract from non-transgenic counterpart cotton; and M, prestained standard markers in kDa
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Fig. 3 Chimeric gene map of the recombinant binary vector: a pBI121BCH carrying the bean Chi gene and nptII gene driven by CaMV35S promoter (P35S). LB left border, RB right border, nptII neomycin phosphotransferase, Chi chitinase, Pnos nopaline synthase promoter, Tnos
nopaline synthase terminator. b pBI121-Cry1Ab carrying the cry1Ab and nptII genes driven by the CaMV35S promoter. LB left border, RB right border, nptII neomycin phosphotransferase, Nos-ter nopaline synthase terminator
Anions analysis
the cotton seeds would represent a potential health benefit and may expand utilization of raw cotton seed (Chapman et al., 2001). To generally reflect the metabolic variations related to transformation and breeding, metabolite profiling might be necessary. According to the results of the present study, the quantity of some metabolites were identified significantly different between transgenic and non-transgenic counterpart, which might be as result of position side effects of the transgene which needs more investigations. Little variations were detected in the fatty acid contents of chitinase line, but these values were within the Crop Composition Database ranges available for cotton seed oil (ILSI Life Sciences Institute) 2006). Hence, the insertion of Bt (Line 61) and chitinase (Line 57) genes to cotton had no effect on the oil quantity and quality of the transgenic lines compared to the non-transgenic counterpart (Table 1). The results reported by Berberich et al. (1996), Nida et al. (1996), and Bertrand et al.(2005) confirmed that the highest quantity of the fatty acids belongs to linoleic acid, palmitic acid, and oleic acid, respectively, which is in accordance to our results. Berberich et al. (1996) did not observe any differences in fatty acid contents of the studied transgenic and non-transgenic cotton seeds, but the results of Nida et al. (1996) showed minor differences for oleic and palmitic acids.
All the major nutrient anions could be easily quantified in the water-extracted samples, with superior sensitivity and reproducibility. Average values determined for aqueous extracts of the studied samples are given in Table 7. The results revealed that there were no statistically significant differences in the levels of the measured anions of transgenic lines comparing to the non-transgenic counterpart. All the anions, including acetate, chloride, nitrate, phosphate, sulfate, succinate, and oxalate in both Bt and chitinase transgenic lines had no considerable variation comparing to the non-transgenic counterpart.
Discussion Transgenes and somaclonal variations related to the regeneration process could be causes of metabolic variations between the transgenic and non-transgenic counterpart (Zhou et al. 2012). Also, the metabolic variations between the transgenic plant and its non-transgenic counterpart might be due to the position side effects of gene inserted; however, these unintended variations might be good. For instance, increased oleic acid content with a compensatory reduction in linoleic acid in
Table 1 Fatty acid composition (%) in cotton seed of Bt, chitinase, and non-transgenic counterpart lines Non-transgenic counterpart
chitinase line
Bt line
ILSI
Myristic acid Palmitic acid Stearic acid Oleic acid
0.3614±0.07b 21.19±0.38a 0.3310±0.18a 18.20±0.85b
0.5612±0.07a 22.08±1.12a 0.1545±0.05b 20.78±0.95a
0.4089±0.05b 21.21±1.09a 0.2354±0.1ab 18.73±1.18b
0.45–2.4* 15.11–27.9 0.2–3.11 12.8–25.3
Linoleic acid
59.91±1.37a
56.38±1.17b
59.41±1.59a
46.0–59.4
Values are presented as mean±SD SD standard deviation Superscript letters indicate fatty acids that show statistically significant differences (P
