Mol. Cells 2018; 41(5): 413-422
Published online May 10, 2018
https://doi.org/10.14348/molcells.2018.2254
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: chungys@dau.ac.kr
Soybean transgenic plants with ectopically expressed
Keywords
Stress conditions, including drought, salinity, and cold, cause severe problems in the production of agricultural crops. Among abiotic stresses, drought stress is one of the most serious limiting factors for plant growth and crop yield. Shortage of water availability leads to a decisive yield losses in most environments (Sinclair et al., 2004; Zhang et al., 2010; Zhu, 2002). Recently, global warming has made a negative impact in agricultural regions. As a climate change limits the water resources for agricultural irrigation, the development of drought tolerant crops has become an important task worldwide. In order to improve the agricultural value under water stress, genetic transformation technology has been used to produce genetically modified (GM) crop varieties for commercial cultivation (Bruce et al., 2002; Li et al., 2017; Xoconostle-Cázares et al., 2010). Plants show a range of adaptations to environmental stresses and use various defense mechanisms to deal with drought stress, including drought escape, drought avoidance, and drought tolerance (Manavalan et al., 2009; Wang et al., 2016; Yang et al., 2011). Abscisic acid (ABA), an important phytohormone, plays an essential role in the adaptive response to abiotic stresses in higher plants during vegetative growth. The level of ABA increases in vegetative tissues and regulates the expression of various stress-responsive genes under stress conditions. When plants are exposed to water deficiency, ABA accumulates in the roots and transfers to leaves through the transpiration stream. ABA concentration increases around guard cells and induces stomatal closure, which minimizes water loss and contributes to plant survival (Choi et al., 2013; Gao et al., 2011; Kang et al., 2002; Lee an Luan, 2012; Luan, 2002; Schroeder et al., 2001; Zeevaart and Creelman, 1988).
ABA-responsive gene expression is regulated by many transcription factors (TFs) that are induced by abiotic stresses and these TFs also mediate defense responses. For example, basic leucine zipper (bZIP) TFs interact with specific ABA-responsive elements (ABREs), which are cis-acting elements containing a (C/T)ACGTGGC consensus sequence that is present in the promoter region of ABA-regulated genes. Therefore, these TFs are termed ABRE-binding factors (ABFs) and the expression of their genes (
Genetic engineering technology offers a possible route to elucidating and overcoming stress effects in plants. Development of genetically modified technology has made a tremendous achievement in solving problems that were difficult to solve with conventional breeding. Advances in genomics have developed commercial cultivars with the utilization of genetic transformation. (Nakashima and Yamaguchi-Shinozaki, 2013; Pathan et al., 2010). Many useful genes have been introduced into soybean using
In the present study, we generated soybean transgenic plants overexpressing
The
Mature soybean seeds of the Korean cultivar ‘Kwangankong’ were used for transformation by following the method described by Kim et al. (2017). Plants with two trifoliate leaves were screened using a herbicide assay to identify transformants that expressed the
Total genomic DNA was extracted from NT (non-transgenic) and transgenic plants using cetyltrimethyl ammonium bromide. A polymerase chain reaction (PCR) analysis was performed to detect
For Southern blot analysis, 10 μg of the genomic DNAs from NT and transgenic plants were digested overnight using
Total RNAs were isolated from NT and transgenic plants using the Plant RNA Purification Reagent (Invitrogen, USA) according to the manufacturer’s instructions. Reverse transcriptase PCR (RT-PCR) was performed using the Maxime RT-PCR Premix (iNtRon, Korea) according to the manufacturer’s instructions. The primers used in the RT-PCR for gene expression were as follows:
Quantitative real-time PCR (qRT-PCR) was performed in 96-well plates with the CFX-96™ Real-Time system (Bio-Rad, Hercules, CA, USA). First-strand cDNA was synthesized using Superscript™ II Reverse Transcriptase (Invitrogen) and oligo-dT (Invitrogen) following the users’ instruction manual. The quantity and quality of the synthesized cDNAs were determined by spectrophotometry. Each reaction contained 3 μl (3 ng/μl) of cDNA, 0.5 μl (10 pm/μl) of each primer and 10 μl SYBR® Premix Ex Taq™ (Takara) in a total reaction volume of 20 μl. The PCR conditions were as follows: 95°C for 3 min; 40 cycles of 10 s at 95°C, 10 s at 55°C, and 20 s at 72°C. A dissociation curve was generated by increasing the temperature from 65 to 95°C to check for amplification specificity. The efficiency and standard deviation of each primer were obtained using Bio-Rad CFX Manager v1. 6. 541. 1028 on a standard curve generated from a two-fold dilution series of one sample at five dilution points for two technical replicates. Baseline and threshold cycle (Ct value) were automatically calculated with default parameters. Expression of
For drought tolerance analysis, NT, EV (transformed with empty vector carrying only
The extent of ion leakage from NT and transgenic plants after stress treatment was investigated by measuring conductivity. Leaf samples (1 g) were soaked in 10 ml of distilled water for 24 h at room temperature and the conductivity of the solution (Lt) was measured using a EC-400L conductivity meter (Istek, Korea). The leaf samples were then returned to the solution in the tubes, which were sealed and incubated at 95°C for 20 min. The solution (L0) was then cooled to room temperature and conductivity was re-measured. The Lt/L0 × 100 values were calculated and used to evaluate relative electrolyte leakage (Fan et al. 1997). Statistical analysis was also performed using the Excel
Total chlorophyll from leaves of NT and transgenic plants after stress treatment was isolated in 80% acetone (v/v). The chlorophyll content was calculated spectrophotometrically as described by Wu (2008). Statistical analysis was also performed using the Excel
NT, EV, and transgenic plants were grown in the same volume of soil and in identical containers in the growth chamber at 25°C, with a long day photoperiod (18 h light/ 6 h dark), and 60% humidity. Leaves at similar developmental stages (fully expanded leaves from 2 nodes) were detached and weighed on a sterile bench in an extractor hood. Leaf weight was measured at 40-min intervals for 200 min and compared to the initial weight. Statistical analysis was performed using the Excel
The behavior of stomatal guard cells was analyzed during water deficit conditions. NT, EV, and transgenic plants were grown in the same volume of soil and in identical containers in the growth chamber at 25°C, with a long day photoperiod (18 h light/6 h dark), and 60% humidity until leaves on 2 nodes were fully expanded. The plants were then subjected to water stress for 20 days. Leaves of similar developmental stage were detached from randomly chosen sites, fixed in 80% acetone, immersed in 5% NaOH, and boiled for 1 min. They were then washed three times with distilled water, and incubated in bleach until they had lost their color (Fu et al., 2002). Photographs were taken with the abaxial side up of leaves from plants after 7, 11, 15, and 20 days of water stress. The number of stomatal guard cells was counted and the stomata were assessed: completely open, partially open, and completely closed (Huang et al., 2009; Choi et al., 2013).
NT and transgenic soybean seeds (T2) were planted in a seedling tray and seedlings were kept in a greenhouse. Plants were watered either regularly (every 4 days) or rarely (every 10 days) to evaluate agronomic traits such as plant height, number of branches per plant, and number of nodes per plant. In addition, the number of pods per plant and the seed weight per plant were counted to investigate the relative yield of transgenic plants. Statistical analysis was performed using the Excel
Recently, stable
Compared with our previous study (Lee et al., 2006), the modified protocol significantly improved production of successful transgenic plants. The modified transformation protocol enabled generation of many different transgenic soybeans; to date, more than 20 different transgenic plants have been produced (data not shown).
To confirm the integration of the transgene in transformed soybean plants, genomic DNA was isolated from T0 transformants and analyzed using PCR for the presence of
The genetic stability of integrated genomic DNA was analyzed by Southern blotting using leaf samples from four T2 seedlings (lines #2, #3, #9, and #10) to determine the copy number of transgenes (Fig. 1C). These four lines were selected because of their phenotype and the production of sufficient seeds. Genomic DNAs from NT, EV, and transgenic plants were digested with
The expression of the transgenes was analyzed by reverse transcriptase-PCR (RT-PCR) using
Overexpression of
The physiological aspects of enhanced drought tolerance in the transgenic lines were analyzed by measuring changes in ion leakage and chlorophyll content. Drought stress resulted in a significant increase in ion leakage from leaves of NT and EV after 11 days of treatment. Transgenic lines #2 and #9 showed increased ion leakage but at a significantly lower level (
Water loss of transgenic plants by transpiration was compared to that from NT and EV plants by weighing leaves immediately after detachment and at intervals over the following 200 min (Fig. 3A). At 200 min after detachment, the water content of NT and EV plant leaves fell to about 55% and 48%, respectively, of the start level. In contrast, those of transgenic lines #2 and #9 decreased to 69% and 72%, respectively (Fig. 3B). Thus, water loss from transgenic lines #2 and #9 was slower than from NT and EV plants (
The ratios of stomatal closure in NT, EV, and transgenic lines #2 and #9 were compared during the drought treatment (Choi et al., 2013; Kang et al., 2002). Stomatal guard cell behavior was analyzed microscopically using detached leaves (data not shown). The degree of stomatal opening was classified as completely open, partially open, or completely closed (Choi et al., 2013; Huang et al., 2009). At the end of the 20-day drought treatment, 3.5% and 56% of the stomata of transgenic lines #2 and #9 were completely closed, respectively. Approximately 44% of stomata in line #2 were completely open, whereas only 16% were completely open in line #9. Almost all stomata were completely open in NT and EV plants. In line #2, 52% of stomata were partially open, whereas only 27% were partially open in line #9 (Fig. 4). Our observations indicate that drought tolerance in
Guard cells are regarded as a good model system for understanding signal transduction in plants. Under a water deficit, plants synthesize ABA, which triggers stomatal closure, even in daytime. Our data confirmed that
Several studies have shown that overexpression of bZIP transcription factor genes enhances the response of transgenic plants to ABA and triggers stomatal closure under stress conditions, resulting in improved tolerance of drought and high salt conditions (Gao et al., 2011; Hossain et al., 2010; Jia et al., 2015; Wang et al., 2016; Yang et al., 2011).
Here, we compared the salt tolerance of NT, EV, and line #2 and #9 plants grown in 200 mM NaCl solution for 11 days and also examined whether expression of
Salt stress, similar to other important abiotic stresses such as drought, cold, and flooding, also inhibits plant growth and crop yield. When plants are exposed to salt stress, extraction of water from the soil to their roots is hampered by the high osmotic pressure in the soil solution. Moreover, plants are also damaged by the accumulation of sodium and chloride (Zhu, 2001; Pathan et al., 2010). This analysis confirmed that drought and salt tolerance was conferred by expression of
To examine the agronomic characteristics of
In this study, we introduced the
Mol. Cells 2018; 41(5): 413-422
Published online May 31, 2018 https://doi.org/10.14348/molcells.2018.2254
Copyright © The Korean Society for Molecular and Cellular Biology.
Hye Jeong Kim1, Hyun Suk Cho1, Jung Hun Pak1, Tackmin Kwon1, Jai-Heon Lee1, Doh-Hoon Kim1, Dong Hee Lee2, Chang-Gi Kim3, and Young-Soo Chung1,*
1Department of Molecular Genetics, College of Natural Resources and Life Science, Dong-A University, Busan 49315, Korea, 2Genomine Advanced Biotechnology Research Institute, Genomine Inc., Pohang 37668, Korea, 3Bio-Evaluation Center, KRIBB, Cheongju 28116, Korea
Correspondence to:*Correspondence: chungys@dau.ac.kr
Soybean transgenic plants with ectopically expressed
Keywords:
Stress conditions, including drought, salinity, and cold, cause severe problems in the production of agricultural crops. Among abiotic stresses, drought stress is one of the most serious limiting factors for plant growth and crop yield. Shortage of water availability leads to a decisive yield losses in most environments (Sinclair et al., 2004; Zhang et al., 2010; Zhu, 2002). Recently, global warming has made a negative impact in agricultural regions. As a climate change limits the water resources for agricultural irrigation, the development of drought tolerant crops has become an important task worldwide. In order to improve the agricultural value under water stress, genetic transformation technology has been used to produce genetically modified (GM) crop varieties for commercial cultivation (Bruce et al., 2002; Li et al., 2017; Xoconostle-Cázares et al., 2010). Plants show a range of adaptations to environmental stresses and use various defense mechanisms to deal with drought stress, including drought escape, drought avoidance, and drought tolerance (Manavalan et al., 2009; Wang et al., 2016; Yang et al., 2011). Abscisic acid (ABA), an important phytohormone, plays an essential role in the adaptive response to abiotic stresses in higher plants during vegetative growth. The level of ABA increases in vegetative tissues and regulates the expression of various stress-responsive genes under stress conditions. When plants are exposed to water deficiency, ABA accumulates in the roots and transfers to leaves through the transpiration stream. ABA concentration increases around guard cells and induces stomatal closure, which minimizes water loss and contributes to plant survival (Choi et al., 2013; Gao et al., 2011; Kang et al., 2002; Lee an Luan, 2012; Luan, 2002; Schroeder et al., 2001; Zeevaart and Creelman, 1988).
ABA-responsive gene expression is regulated by many transcription factors (TFs) that are induced by abiotic stresses and these TFs also mediate defense responses. For example, basic leucine zipper (bZIP) TFs interact with specific ABA-responsive elements (ABREs), which are cis-acting elements containing a (C/T)ACGTGGC consensus sequence that is present in the promoter region of ABA-regulated genes. Therefore, these TFs are termed ABRE-binding factors (ABFs) and the expression of their genes (
Genetic engineering technology offers a possible route to elucidating and overcoming stress effects in plants. Development of genetically modified technology has made a tremendous achievement in solving problems that were difficult to solve with conventional breeding. Advances in genomics have developed commercial cultivars with the utilization of genetic transformation. (Nakashima and Yamaguchi-Shinozaki, 2013; Pathan et al., 2010). Many useful genes have been introduced into soybean using
In the present study, we generated soybean transgenic plants overexpressing
The
Mature soybean seeds of the Korean cultivar ‘Kwangankong’ were used for transformation by following the method described by Kim et al. (2017). Plants with two trifoliate leaves were screened using a herbicide assay to identify transformants that expressed the
Total genomic DNA was extracted from NT (non-transgenic) and transgenic plants using cetyltrimethyl ammonium bromide. A polymerase chain reaction (PCR) analysis was performed to detect
For Southern blot analysis, 10 μg of the genomic DNAs from NT and transgenic plants were digested overnight using
Total RNAs were isolated from NT and transgenic plants using the Plant RNA Purification Reagent (Invitrogen, USA) according to the manufacturer’s instructions. Reverse transcriptase PCR (RT-PCR) was performed using the Maxime RT-PCR Premix (iNtRon, Korea) according to the manufacturer’s instructions. The primers used in the RT-PCR for gene expression were as follows:
Quantitative real-time PCR (qRT-PCR) was performed in 96-well plates with the CFX-96™ Real-Time system (Bio-Rad, Hercules, CA, USA). First-strand cDNA was synthesized using Superscript™ II Reverse Transcriptase (Invitrogen) and oligo-dT (Invitrogen) following the users’ instruction manual. The quantity and quality of the synthesized cDNAs were determined by spectrophotometry. Each reaction contained 3 μl (3 ng/μl) of cDNA, 0.5 μl (10 pm/μl) of each primer and 10 μl SYBR® Premix Ex Taq™ (Takara) in a total reaction volume of 20 μl. The PCR conditions were as follows: 95°C for 3 min; 40 cycles of 10 s at 95°C, 10 s at 55°C, and 20 s at 72°C. A dissociation curve was generated by increasing the temperature from 65 to 95°C to check for amplification specificity. The efficiency and standard deviation of each primer were obtained using Bio-Rad CFX Manager v1. 6. 541. 1028 on a standard curve generated from a two-fold dilution series of one sample at five dilution points for two technical replicates. Baseline and threshold cycle (Ct value) were automatically calculated with default parameters. Expression of
For drought tolerance analysis, NT, EV (transformed with empty vector carrying only
The extent of ion leakage from NT and transgenic plants after stress treatment was investigated by measuring conductivity. Leaf samples (1 g) were soaked in 10 ml of distilled water for 24 h at room temperature and the conductivity of the solution (Lt) was measured using a EC-400L conductivity meter (Istek, Korea). The leaf samples were then returned to the solution in the tubes, which were sealed and incubated at 95°C for 20 min. The solution (L0) was then cooled to room temperature and conductivity was re-measured. The Lt/L0 × 100 values were calculated and used to evaluate relative electrolyte leakage (Fan et al. 1997). Statistical analysis was also performed using the Excel
Total chlorophyll from leaves of NT and transgenic plants after stress treatment was isolated in 80% acetone (v/v). The chlorophyll content was calculated spectrophotometrically as described by Wu (2008). Statistical analysis was also performed using the Excel
NT, EV, and transgenic plants were grown in the same volume of soil and in identical containers in the growth chamber at 25°C, with a long day photoperiod (18 h light/ 6 h dark), and 60% humidity. Leaves at similar developmental stages (fully expanded leaves from 2 nodes) were detached and weighed on a sterile bench in an extractor hood. Leaf weight was measured at 40-min intervals for 200 min and compared to the initial weight. Statistical analysis was performed using the Excel
The behavior of stomatal guard cells was analyzed during water deficit conditions. NT, EV, and transgenic plants were grown in the same volume of soil and in identical containers in the growth chamber at 25°C, with a long day photoperiod (18 h light/6 h dark), and 60% humidity until leaves on 2 nodes were fully expanded. The plants were then subjected to water stress for 20 days. Leaves of similar developmental stage were detached from randomly chosen sites, fixed in 80% acetone, immersed in 5% NaOH, and boiled for 1 min. They were then washed three times with distilled water, and incubated in bleach until they had lost their color (Fu et al., 2002). Photographs were taken with the abaxial side up of leaves from plants after 7, 11, 15, and 20 days of water stress. The number of stomatal guard cells was counted and the stomata were assessed: completely open, partially open, and completely closed (Huang et al., 2009; Choi et al., 2013).
NT and transgenic soybean seeds (T2) were planted in a seedling tray and seedlings were kept in a greenhouse. Plants were watered either regularly (every 4 days) or rarely (every 10 days) to evaluate agronomic traits such as plant height, number of branches per plant, and number of nodes per plant. In addition, the number of pods per plant and the seed weight per plant were counted to investigate the relative yield of transgenic plants. Statistical analysis was performed using the Excel
Recently, stable
Compared with our previous study (Lee et al., 2006), the modified protocol significantly improved production of successful transgenic plants. The modified transformation protocol enabled generation of many different transgenic soybeans; to date, more than 20 different transgenic plants have been produced (data not shown).
To confirm the integration of the transgene in transformed soybean plants, genomic DNA was isolated from T0 transformants and analyzed using PCR for the presence of
The genetic stability of integrated genomic DNA was analyzed by Southern blotting using leaf samples from four T2 seedlings (lines #2, #3, #9, and #10) to determine the copy number of transgenes (Fig. 1C). These four lines were selected because of their phenotype and the production of sufficient seeds. Genomic DNAs from NT, EV, and transgenic plants were digested with
The expression of the transgenes was analyzed by reverse transcriptase-PCR (RT-PCR) using
Overexpression of
The physiological aspects of enhanced drought tolerance in the transgenic lines were analyzed by measuring changes in ion leakage and chlorophyll content. Drought stress resulted in a significant increase in ion leakage from leaves of NT and EV after 11 days of treatment. Transgenic lines #2 and #9 showed increased ion leakage but at a significantly lower level (
Water loss of transgenic plants by transpiration was compared to that from NT and EV plants by weighing leaves immediately after detachment and at intervals over the following 200 min (Fig. 3A). At 200 min after detachment, the water content of NT and EV plant leaves fell to about 55% and 48%, respectively, of the start level. In contrast, those of transgenic lines #2 and #9 decreased to 69% and 72%, respectively (Fig. 3B). Thus, water loss from transgenic lines #2 and #9 was slower than from NT and EV plants (
The ratios of stomatal closure in NT, EV, and transgenic lines #2 and #9 were compared during the drought treatment (Choi et al., 2013; Kang et al., 2002). Stomatal guard cell behavior was analyzed microscopically using detached leaves (data not shown). The degree of stomatal opening was classified as completely open, partially open, or completely closed (Choi et al., 2013; Huang et al., 2009). At the end of the 20-day drought treatment, 3.5% and 56% of the stomata of transgenic lines #2 and #9 were completely closed, respectively. Approximately 44% of stomata in line #2 were completely open, whereas only 16% were completely open in line #9. Almost all stomata were completely open in NT and EV plants. In line #2, 52% of stomata were partially open, whereas only 27% were partially open in line #9 (Fig. 4). Our observations indicate that drought tolerance in
Guard cells are regarded as a good model system for understanding signal transduction in plants. Under a water deficit, plants synthesize ABA, which triggers stomatal closure, even in daytime. Our data confirmed that
Several studies have shown that overexpression of bZIP transcription factor genes enhances the response of transgenic plants to ABA and triggers stomatal closure under stress conditions, resulting in improved tolerance of drought and high salt conditions (Gao et al., 2011; Hossain et al., 2010; Jia et al., 2015; Wang et al., 2016; Yang et al., 2011).
Here, we compared the salt tolerance of NT, EV, and line #2 and #9 plants grown in 200 mM NaCl solution for 11 days and also examined whether expression of
Salt stress, similar to other important abiotic stresses such as drought, cold, and flooding, also inhibits plant growth and crop yield. When plants are exposed to salt stress, extraction of water from the soil to their roots is hampered by the high osmotic pressure in the soil solution. Moreover, plants are also damaged by the accumulation of sodium and chloride (Zhu, 2001; Pathan et al., 2010). This analysis confirmed that drought and salt tolerance was conferred by expression of
To examine the agronomic characteristics of
In this study, we introduced the
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