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The latest report on the draft genome of Brassica rapa sequence has been published. To elucidate the functions of these genes and to efficiently search for agriculturally useful genes, a Full-length cDNA Over-eXpressor (FOX) gene hunting system was used. The FOX library from Chinese cabbage was introduced into rice via Agrobacterium-mediated transformation. Approximately 1,150 FOX-rice lines were generated. Genomic PCR analysis indicated that the average length of FL-cDNAs introduced into individual lines was 900~1,200 bp. Basic Local Alignment System Tool (BLAST) analysis of the FL-cDNA genes revealed that 35.5% have unknown function. Most of the randomly selected transgenic rice lines showed overexpression (92%) of these genes relative to the wild-type Gopum. Moreover, 94% of the 850 transgenic rice lines were moderately tolerant (slightly yellow) to cold and 9 lines were tolerant (seedlings were light green). Morphological evaluation of the transgenic rice lines showed minimal phenotypic alteration (12%). Approximately 25.1% and 22% of the plants were significantly ahead in the days to heading and had elevated chlorophyll content, respectively. Other agronomic traits such as filled grains, number of tiller, panicle length, and culm and plant height were relatively less variable among the transgenic lines. These results provide a resource for defining genes that are associated with tolerance in transgenic rice lines.
Chinese cabbage (Brassica rapa subsp. pekinensis) is one of the most important Brassica species vegetables found in Asian countries. The draft genome sequence of B. rapa has been recently published (The Brassica rapa Genome Sequencing Project Consortium 2011). The Chinese cabbage genome contains 41,174 protein-coding regions, whereas 27,379 genes are currently believed to exist in the Arabidopsis genome (TAIR9 information: http://www.arabidopsis.org/). Appropriate biological experiments are needed to uncover the functions of these genes.
To accelerate the identification and characterization of gene function, various resources, including Arabidopsis and rice information, are publicly available (Nakamura et al. 2007). The Arabidopsis Information Resource (TAIR) maintains a database of genetic and molecular biology data for the model higher plant Arabidopsis thaliana (www.arabidopsis.org). Data available from TAIR includes the complete genome sequence along with gene structure, gene product information, metabolism, gene expression, DNA and seed stocks, genome maps, genetic and physical markers, publications, and information about the Arabidopsis research community. Along with this, infrastructure for rice bioin-formatics (reviewed in Sasaki et al. 2005), including integrated databases such as RAP-DB (Ohyanagi et al. 2006; Rice Annotation Project 2007) and the TIGR Rice Genome Annotation Database (Ouyang et al. 2007), BGI- RIS (Zhao et al. 2004), and MOsDB (Karlowski et al. 2003), is also publicly available. The available information provides an important resource for the analysis of Brassica rapa genes.
For large-scale identification of gene function, several systems and tools have been developed to generate loss- of-function mutants (Hirochika et al. 2004; An et al. 2005). These loss-of-function resources and databases are very important for tagging and hunting non-redundant genes in the rice genome. However, 29% of rice genes are estimated to exist within clustered and redundant gene families (IRGSP 2005). Therefore, it is difficult to find loss-of- function phenotypes for these duplicated genes. Moreover, when knockouts of genes confer embryonic lethality or severe developmental defects upon plants, it is impossible to the phenotypes associated with mutation or deletion of these genes (Nakamura et al. 2007). To solve this problem, FL-cDNA clones (Seki et al. 2002; Ichikawa et al. 2006) were recently developed as an alternative gain-of-function approach for the systematic elucidation of gene functions in Arabidopsis; the system is the so-called Full-length cDNA Over-eXpressor gene hunting system (FOX hunting system). FOX uses ectopic overexpression of a single or limited numbers of FL-cDNA(s) in individual transgenic plants. It generates large numbers of dominant mutations, enabling comprehensive characterization of novel and important traits and identification of the causal genes.
Studies on genetic transformation of plants have revealed an unexpected finding; the loss of mRNA encoded by either the introduced transgene or the transgene and endogenous homologous gene(s) (Vaucheret and Fagard 2001). These gene-silencing phenomena are believed to be due to methylation induced premeiotically (MIP), repeat- induced point mutation (RIP), RNA interference (RNAi), and repeat-induced gene silencing (RIGS) (Fagard and Vaucheret 2000; Marathe et al. 2000). In order to avoid this problem during our study of gene function, FL-cDNAs genes from Chinese cabbage were transformed into the rice cultivar Gopum.
In this study, clones with full-length cDNA from Chinese cabbage were sequenced, and transgenic rice plants were subsequently generated via Agrobacterium- mediated transformation. The function of each clone was classified according to the annotation function based on BLAST analysis (National Center for Biotechnology Information; www.ncbi.nlm.gov). Moreover, the FOX rice lines generated were evaluated in the fields to determine their morpho-agronomic characteristics and phenotypic traits, as well as their responses to cold and salinity.
MATERIALS AND METHODS
Plant materials and field trials
The rice variety Gopum was used as the wild-type strain in the generation of transgenic rice. Regenerated plants were transplanted in soil consisting of 50% compost and 50% earth soil and acclimatized for 2 weeks in a greenhouse. Young leaves were collected for genomic DNA analysis using pBigs_sfl1 and hpt primers (GenBank Accession No. AF234296). Subsequently, plants that were confirmed to carry the gene insert were harvested, and the T1 seeds were used in the succeeding generation in the field following the protocol of Sun et al. (2011). The varieties were sown in a seedling-growing tray and placed in the greenhouse for 3 weeks in May, 2011. Grown seedlings were transplanted in the experimental farm spaced at 30×15 cm with one seedling per hill and arranged in randomized complete block design replicated three times. The cultivation and management were performed according to the standards of the experimental farm in Chungbuk National University.
Vector construction with full-length cDNAs
Brassica rapa clones with full-length cDNA were ligated into the pBigs_sfiI vector. The recombinant vector carrying these clones was constructed under the control of the pUBI-1 promoter and NOS terminator, as shown in Fig. 1. Each clone was grown in Luria-Bertani medium containing 50 mg/L kanamycin sulfate and incubated at 28°C on a shaker for 24~36 h. Plasmid DNA, extracted according to the Qiagen plasmid kit (Cat. No. 12125) protocol, was used in the sequence analysis.
Generation of transgenic rice
Transgenic rice was generated following the protocol of Toki et al. (2006), with some modifications (Lee et al. 2011). Briefly, dehulled mature seeds of the japonica rice cultivar Gopum were sterilized with 70% ethanol for 5 min. These seeds were further sterilized by strong shaking in 30–35 mL of 2.5% sodium hypochlorite containing one drop of Tween 20 for 15 min. The sterilized seeds were washed 20 times in distilled water, dried in the clean bench for 30 min, and then inoculated on embryogenic callus induction (2N6) medium (Table 1). This set up was incubated in the dark at 32°C for 7–10 days.
Agrobacterium strain EHA105 harboring binary Ti plasmid pFLCIII (Fig. 1) containing the full- length cDNA gene was cultured on AB medium containing 50 mg/L kanamycin sulfate solidified with 1.5% agar incubated for 3 days at 28°C in the dark. Agrobacterium culture was scraped from the plates and suspended in AAM medium to yield an OD600 of approximately 0.3. Pre-cultured seeds were immersed in the Agrobacterium suspension by gently inverting the tube for 15 min, blotted dry with a sterilized filter paper to remove excess bacteria. The seeds were transferred onto a sterilized filter paper that had been moistened with 0.5 mL of AAM medium placed on 2N6-AS medium solidified with 0.4 % Gelrite. After 3 days of co-cultivation at 25°C in the dark, seeds were washed 5 times in sterilized water, and then washed once in sterilized water containing 500 mg/L carbenicillin to remove Agrobacterium cells. The seeds were rapidly blotted dry on a sterilized filter paper and cultured on N6D (Table 2) medium containing 50 mg/L hygromycin and 400 mg/L carbenicillin under continuous light at 32°C for 2 weeks. Proliferating calli arising from the scutellum were transferred to REIII (Table 2) medium. Plantlets arising from the calli were transferred to HF medium to induce root formation.
DNA extraction and PCR analysis
Genomic DNA was extracted as described by Cho et al. (2007) with some modifications. The relative purity and concentration of extracted DNA was estimated using NanoDrop-1000 (NanoDrop Technologies, Inc. USA). PCR analyses were performed using HPT-Fw (5′-GGA-TTTCGGCTCCAATGTCCTGACGGA-3′) and HPT-Rv (5′-CTTCTACACAGCCATCGGTCCAGA-3′) primers to check the introgression of hygromycin phosphotransferase gene (hpt) and pBigs_SfiI-Fw (5′-TATTCGGAGAGGG-TACGTATTTTTAC-3′) and pBigs_sfiI-Rv (5′-GCA-ACAGGATTCAATCTTAAGAAACT-3′) to check the introduction of the full-length cDNA gene.
Southern blot analysis
For DNA blot analysis, 10 μg of genomic DNA was extracted as mentioned above. A digoxigenin (DIG) probe was synthesized according to the manufacturer’s instructions (PCR DIG Probe Synthesis Kit; Roche Molecular Biochemicals, USA). The template for the probe synthesis was from the vector containing the full-length cDNA clone. The forward/ reverse primers used for the synthesis of the probe were 5′-GATTAAGCATCCAAATGTTGTTCA-3′ and 5′-CCTACGAGCCTCATCTTCT-3′. The PCR amplification was initiated with a denaturation at 95°C for 2 min, followed by 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 60 s, and the final elongation step at 72°C for 7 min. Ten micrograms of genomic DNA was digested overnight at 37°C with the restriction enzymes BamHI and EcoRI. The digested DNA was subjected to electrophoresis in a 0.8% agarose gel and transferred to a Hybond-N+ membrane (Amersham Biosciences, USA), according to the manufacturer’s instructions. The membrane was pre- hybridized at 42°C in a DIG East Hyb buffer for 1 h. A DIG-labeled probe was then added (2 μL of probe/mL of buffer) and hybridization was carried out overnight at 42°C. The membrane was washed twice with a low- stringency washing solution (2× SSC, 0.1% SDS) at 25°C for 5 min, and then washed twice with a high-stringency solution (0.1× SSC, 0.5% SDS) at 68°C for 15 min. The DIG-labeled DNA was detected according to the manufacturer’s instructions (DIG Nucleic Acid Detection Kit; Roche Molecular Biochemicals, USA).
Cold, salinity, and drought tolerance screen
Cold evaluation was conducted at the Chuncheon Experimental Station of the Rural Development Administration, Korea. About 50 seeds were directly seeded into germination trays (60 × 30 × 3.5 cm) with a soil-free medium of steam-sterilized sand with peat compost and vermiculite and allowed to grow for 7 weeks before cold treatment at a water temperature of approximately 13°C, and an air temperature of 30–35°C.
For the salinity and drought tolerance test, styrofoam seedling floats were fabricated as described by Manigbas et al. (2011). Holes were drilled at the surface of the styrofoam with a nylon net supporting the bottom. Two pre-germinated seeds were placed on each hole. Seedling floats were placed on each tray comprising a replication of two, with 20 seedlings per entry. The growing plants were supplied with Murashige and Skoog (MS) solution at 4.4 gram per liter to maintain good growth and healthy seedlings throughout the experiment. After 2 weeks, solutions of 130 mM NaCl and 20% PEG were used to induce salinity and drought, respectively on rice seedlings; this solution was changed after 8 days. The lines were scored after 2 weeks, or when the susceptible check showed high susceptibility by using the standard evaluation system for rice (IRRI-SES 2007).
RT-PCR analysis
Total RNA was extracted from the transgenic lines and wild-type Gopum plant by using the RNeasy Plant Mini Kit (QIAGEN, Maryland, USA), according to the manufacturer’s instructions. This process was repeated in all experiments requiring RNA extraction. The relative purity and concentration of extracted RNA was estimated using NanoDrop-1000 spectrophotometer (NanoDrop Technologies, Inc. USA), and stored in a −80°C freezer. Total RNAs were cleaned using DNaseI, and first-strand cDNA synthesis was performed by reverse transcription of mRNA using Oligo (dT)20 primer and SuperScript™ III Reverse Transcriptase.
The nucleotide sequence of each of the randomly selected clones was used to design primer pairs (Table 3). The specific sequences of the primer pairs were used in a semi-quantitative reverse transcription PCR (RT-PCR). Actin primers were used as loading controls and used as internal controls for normalization of the quantitative RT-PCR reaction.
Statistical analysis
Data requiring statistical analysis were computed using the Statistix version 8 (www.statistix.com). Significant P values were analyzed by using the two-sided Dunnett’s multiple comparison, with the wild-type Gopum as control by using the same software. The frequency distribution of various agronomic traits and sequences was determined using Microsoft Excel 2007.
Nucleotide sequence analysis
All the inserted sequences were checked using the BLAST program in NCBI sequence database and another program in the GenBank/EMBL/DDBJ sequence database. The open reading frame (ORF) and conserved domain were generated using the BLASTN program (http://www.ncbi.nlm.nih.gov/). Sequence alignment, ORF translation, molecular weight calculation of the predicted proteins, and structural analysis of the deduced proteins were carried out by DNAStar’s Lasergene sequence analysis software (www.dnastar.com).
RESULTS
Characterization of selected clones from FOX hunting system
Agrobacterium clones containing FL-cDNAs genes from Brassica rapa were subjected to genomic PCR using forward and reverse primers (pBigs_SfiI-Fw and pBigs_ sfiI-Rv) derived from the flanking sequences. Of the 933 clones analyzed, 80% showed single bands, while 4% showed multiple fragments (Fig. 2A). The remaining clones (16%) did not undergo fragment amplification. All the amplified single PCR fragments bearing the integrated FL-cDNA(s) from B. rapa clones were used to generate their respective sequences. The sizes of the introduced FL-cDNAs were variable, ranging from 100 to 2,400 bp (Fig. 2B). About 5% of the clones were smaller than 500 bp, whereas 95% ranged from 600 to 2,400 bp.
The Basic Alignment System Tool (BLAST) provided by the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) was used to determine the functional annotation of each clone. The frequency distribution based on function is presented in Fig. 2C. Variable gene function was observed from the viewpoint of molecular as well as biological importance. Most clones (32.15%) were characterized as having an unknown biological/molecular gene function. About 0.64% of clones appeared to possess functions related to catalytic activity, and 5.04% appeared to exhibit some binding activity. Transcription- and translation-related functions accounted for 2.36% of the clones. Moreover, 1.39% of the clones had homology with zinc finger-related proteins. A large number of other functions, each with a lower frequency than those reported above, collectively accounted for another 35.16% of the clones (Fig. 2C). These genes provide important resources for studying their function in plants.
Generation of transgenic rice lines
Using the protocol described by Lee et al. (2011) and modified on the basis of that described by Toki et al. (2006), transgenic rice plantlets were regenerated within a month starting from the aseptic culture of the mature seeds (Fig. 3A). The plantlets were subjected to PCR analysis to verify the integration of FL-cDNAs. The genomic DNA PCR analysis is represented in Fig. 3B. Most of the regenerated plants (T0) (73%) contained the expected band sizes (Fig. 3C), indicating the high efficiency of the transformation method. About 20% of the transgenic plants had hpt gene, whereas 2% had only FL-cDNAs gene.
The confirmed FOX rice lines were advanced in the succeeding planting season from 2009 to 2011. So far, we have propagated transgenic rice up to the fourth generation (T4) (Table 4). Most of the transgenic rice lines were in the T1 generation that constituted 470 independent rice lines in 137 independent clones. In the T2 generation, 408 lines with 88 gene inserts were produced. In the T3 generation, 46 lines were generated. In summary, a total of 250 independent clones were inserted into the rice genome, leading to the generation of 1,150 independent transgenic lines (Table 4). These transgenic rice lines are a valuable resource for future analyses designed to determine the functions of each gene.
Gene copy number
To examine the gene copy number of 31 randomly selected lines, we used the digoxigenin (DIG) system method (Roche Molecular Biochemicals, USA). Because of variability in the gene sequences, we used hpt gene as probe. Our results showed that most of the transgenic lines have a single copy number (Fig. 4A). However, in BR 37, which encodes CBL-interacting protein kinase 1, two gene copies were present in each of 4 lines (numbers 3, 4, 6, 7). Similarly, in BR 15, which encodes UDP-glucose-4- epimerase 1, two lines had two copies of the gene (numbers 12 and 13). Furthermore, in clone BR 4, two copies of the introduced gene (actin-depolymerizing factor 2) were observed. The rest of the lines harbored transgenes at single copy. The variability in copy number has been reported in previous studies (Schrider and Hahn 2010). This may be due to the positional effect of the gene during transformation (Gelvin 2003).
Expression analysis of transgenes
To examine whether the expression of transgenes was elevated at the molecular level, 24 transgenic lines encoding different gene inserts were randomly selected. Expression of these 24 genes was examined by semi- quantitative RT-PCR analysis with individual gene-specific primers (as shown in Table 3) by using the young shoots of each rice seedling. mRNA expression was observed in all 24 genes with varying band intensities (Fig. 4B). Strong mRNA expression was observed in BR 2 (cysteine protease 1), BR 7 (DEAD box RNA helicase RH15), BR 37 (CBL-interacting protein kinase 1), BR 47 (unknown gene function), BR 74 (haloacid dehalogenase- like hydrolase), BR 192 (glycosyltransferase), BR 15 (UDP-glucose-4- epimerase 1), BR 68 (unknown function), and BR 22 (hydrogen-transporting ATPase). Moreover, weak mRNA expression was observed in BR 4 and BR 23 lines encoding an actin-binding/depolymerizing factor 2 and endoplasmic reticulum auxin-binding protein 2 mRNA, respectively.
Response of transgenic rice lines against cold and salinity
To determine the response of transgenic rice lines to cold, 840 independent lines comprising 250 genes were evaluated at the Chuncheon Experimental Station of the Rural Development Administration, Korea. Most of the transgenic rice lines (94%) were moderately tolerant (seedlings were light yellow) to cold and about 5% were moderately susceptible (seedlings were turning brown) (Fig. 5A). Interestingly, 9 (1%) of the transgenic lines were tolerant (seedlings were light green) (Table 1). Of the 9 tolerant lines, 4 encode genes with an unknown function (BR 5, BR 119, BR 522, BR 503). Other tolerant lines encoded UDP-galactose transporter 3 (BR 457), DEAD box RNA helicase RH 15 (BR 7), Arv1-like protein (BR 115), carbon-monooxide oxygenase (ACYB-1) (BR 39) and light harvesting complex PSII (BR 541). Conversely, we found 6 transgenic lines that were susceptible to cold stress, 4 of which encode an unknown gene function. The rest of the transgenic lines responded to the cold in a manner indistinguishable from wild-type Gopum.
In the salinity test, 85% of the transgenic rice lines were found to be highly susceptible (almost all plants were dead or dying) (Fig. 5B). However, we found 7 tolerant transgenic lines (nearly normal growth and tillering) (Table 1). These lines were BR 7 (unknown gene function), BR 37 (CBL-interacting protein kinase 1), BR 40 (Phosphatase 2C), BR 74 (Haloacid dehalogenase-like hydrolase), BR 91 (unknown gene function), BR 115 (BREB subfamily A-4 ERF/AP2 transcription factor), and BR 119 (unknown gene function). Moreover, 15 lines were shown to be moderately tolerant to salinity stress (their growth was nearly normal, but plants had some rolled leaves). These tolerant lines are an important resource for studying the mechanisms of resistance, and may eventually facilitate crop improvement in the future.
Phenotypic characteristics of the FOX rice lines
To determine the phenotype of the FOX rice lines, all developed lines were evaluated in the field arranged in an incomplete block design. As shown in Fig. 6, the variability in the number of transgenic lines was significant in each of the agronomic traits measured. Indeed, 25.1% matured significantly earlier and 22% contained significantly higher chlorophyll content. Furthermore, 18% of the transgenic rice lines had lower chlorophyll contents. Other agronomic traits such as filled grains, number of tiller, panicle length, and culm length and plant height showed relatively low variability among lines. All morpho- agronomic traits are recorded. For example, the BR 4 line, which encodes actin-depolymerizing factor 2, exhibited a higher number of tillers. Similarly, BR 119, which encodes a gene with unknown function, gave rise to shorter plants.
The altered agronomic traits observed in the FOX lines may be caused by each gene. Further functional analysis is necessary to determine the function of each gene. Regardless of this, these results provide the basic information required to accelerate future functional studies.
DISCUSSION
A total of 1,150 transgenic rice lines with FL-length cDNA genes from Chinese cabbage were generated; these lines contained 250 Brassica rapa FL-cDNAs (Table 4). The size distribution pattern and the average size of FL-cDNAs inserted into the genomes of 250 FOX-rice lines was 900 to 2,100 bp (Fig. 2B). Analyses of individual gene inserts revealed that genes associated with tolerance to cold and salinity were functionally diverse, both at the molecular and biological levels (Fig. 2C). Approximately 1/3 of the gene inserts have no known ascribed function. Furthermore, most of the randomly selected transgenic lines were present as single gene copies (Fig. 3). We overexpressed a variety of transgenes in transgenic rice (Fig. 4). We suggest that our approach will be useful for systematic genome-wide gain-of-function analyses of the Brassica rapa genes that have been inserted into rice.
Phenotypic databases of rice properties have been developed by several groups. These include the International Rice Information System (IRIS) (Wu et al. 2005), Oryzabase (Kurata and Yamazaki 2006), Rice Mutant Database (RMD; Zhang et al. 2006), Rice Tos17 Insertion Mutant Database (Miyao et al. 2007), and Gramene (http://www.gramene.org/). In addition to these useful databases, the transgenic rice lines harboring FL-cDNAs gene from Brassica spp. are expected to provide valuable information for researchers to uncover functions of Brassica genes. Appropriate biological or molecular methodologies are needed to reveal the function of each gene.
We found that 7.9% of the observed transgenic rice plants showed morpho-agronomically altered phenotypes (Fig. 6). Among the 1,150 transgenic lines, 224 lines (26.4%) had a significantly altered number of days to heading. Similarly, 40% had a significant change in the chlorophyll content. Examples of these altered phenotypes are presented in Fig. 7. This rate was much higher than that determined by using activation tagging of rice populations (Jeong et al. 2002). However, for culm length, plant height, panicle length, number of tillers/hill, and percent filled spikelet/panicle, only 3.7% of transgenic lines were different compared to the wild type. During Agrobacterium transformation, minimal weak growth of the regenerants was observed. This is consistent with a previous report (Nakamura et al. 2007) and underscores the notion that transgenic regenerants often suffer from the effects of environmental factors such as humidity, temperature, macro- and micro-nutrient deficiencies or excesses, and microbes (pathogens), especially in the initial growth stage after transfer to soil. Alternatively, during tissue culture for selection and regeneration of transgenic plants, a variety of both epigenetic (e.g., DNA methylation, gene silencing, activation of retrotransposons and transposable elements) and genetic (e.g., nucleotide substitutions, deletions, insertions, rearrangements) alterations may occur in the plant genome (Kaeppler et al. 2000; Cheng et al. 2006; Noro et al. 2007). These factors, i.e., abiotic and biotic factors and tissue culture-induced variations, may influence the growth conditions to some degree, and consequently, alter the phenotypes of transgenic plants. This may explain the increased rate of phenotype alteration in our transgenic lines (Nakamura et al. 2007).
The response of transgenic rice lines to salinity and cold was tested on 900 independent lines. Eight hundred forty six (94%) of the lines were moderately tolerant to the cold stress (Fig. 5A), and 9 lines (1%) were tolerant. This is not surprising since wild-type Gopum is moderately tolerant to cold stress (Choi et al. 2007). However, most of the transgenic lines (85%) were highly susceptible to salinity stress (Fig. 5B). Only 5% were moderately tolerant, and 2% were tolerant in the transgenic rice lines. In any case, our results have identified possibly agronomically important genes that should be characterized in future studies. Furthermore, manipulation of these genes may lead to improvements in the tolerance parameters of this economically important crop.
The development of full-length cDNA collections is an effective strategy for increasing the catalog of gene transcripts. These data serve as a valuable resource for describing gene expression profiles, and ultimately classify genes into families based on their functions (Abe et al. 2011). Full-length cDNA can also serve as a powerful tool to facilitate genomic or other research (Seki and Shinozaki 2009). Several techniques have been established to prepare enriched full-length cDNA libraries (Seki et al. 1998). The utility of full-length cDNAs has been confirmed in various plants, including Arabidopsis (Yang et al. 2006; Alexandrov et al. 2006), rice (Ralph et al. 2008), poplar (Jia et al. 2006), maize (Alexandrov et al. 2009), tomato (Town et al. 2006), and soybean (Umezawa et al. 2008). A major advantage of this approach is that most of the clones contain the complete coding sequence, in addition to the 5′- and 3′-untranslated regions.
In this study, we have described the phenotypes and abiotic reactions of individual transgenic rice lines that carry gene inserts isolated from Chinese cabbage. Our results have thus provided a list of potentially economically important genes that can be further studied during future abiotic studies and functional analyses.
This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ008529), Rural Development Administration and the research grant of the iPET, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
Fig. 1
Schematic diagram of the binary Ti plasmid pFLCIII containing the full length cDNA from Chinese cabbage. pBigs vector was consisted of two different SfiI sites [SfiI(A) and SfiI(B)].
Fig. 2
PCR amplification and BLAST analyses of 933 clones. (A) Pattern and numbers of cDNA-containing fragments. (B) frequency of gene according to the length (bp) of FOX cDNA library. (C) NCBI database annotation based on gene function.
Fig. 3
Development, PCR analysis and transformation efficiency in the generation of transgenic rice. (A) Development of selected 250 clones from FL-cDNA through Agrobacterium-mediated transformation (A) callus formation; (B) inoculation; (C) callus selection using hygromycin (D), (E) and (F) shoot formation and elongation; (G) and (H) root formation and elongation; (I) acclimatization; (B) Genomic PCR confirmation of inserted genes using T1 plants by pBigs SfiI (a) and HPT (b) primers in 1% agarose gel. L= Ladder,1 = BR2, 2=BR4, 3=BR7, 4=BR23, 5=BR47, 6=BR61, 7=BR74, 8=BR91, 9=BR129, 10=BR192, 11=BR195, 12=BR174, 13=BR15, 14=BR37, WT= wild type; (C) The efficiency of Agrobacterium-mediated transformation based on the genomic PCR analysis of 1,072 T0 generation rice lines using hpt and pBigs_SfiI primers.
Fig. 4
Southern blot analysis and expression pattern of randomly selected FOX rice lines. (A) hpt gene was used as probe using a non-isotope method (dioxiginin). BR 37 (BrCIPK1, CBL-interacting protein kinase 1); BR15 (BrUGE1, UDP-glucose-4-epimerase 1); BR2 (BrCP1, Cysteine protease 1); BR4 (Actin depolymerizing factor 2); BR119 (Unknown function); BR23 (ER auxin-binding protein 2); BR29 (Phospholipase C). (B) RT-PCR of selected rice lines using their respective primer pairs (Supplemental Table 2. Rice actin was used as loading adjustments (data not shown). Gopum was the wild type.
Fig. 5
Response of FOX rice lines against cold and salinity (see materials and methods for details).
Fig. 6
Morpho-agronomic variability of 1,150 transgenic rice lines. Each transgenic rice line was compared to the wild type Gopum to generate significant differences (positive/negative) by two-pair wise Dunnet’s.
Fig. 7
The field evaluation of transgenic rice. A to C, the field plots of the transgenic lines in the vegetative stage arranged in incomplete block design; D, high senescense observed in BR 73; E, mild senescence in BR61; F, observed dwarf in BR122; G, partial sterility observed in BR 452.
Table 1
Identified transgenic rice with cold, salinity and drought tolerance.
Table 1
Clone
Tolerance to NaCl stresses (130 mM)
Tolerance to Cold
Drought (20% PEG)
Gene function (based on BLAST)
BR5
−
++
−
Unknown function
BR7
+
++
+
DEAD BOX RNA helicase RH15
BR15
−
+
+
UDP-glucose 4-epimerase
BR37
+
++
+
CBL-interacting protein kinase 1
BR39
−
++
−
Carbon-monoxide oxygenase (ACYB-1)
BR40
+
+
−
Phosphatase 2C, putative/PP2C
BR74
+
+
−
Haloacid dehydrogenase-like hydrolase
BR91
+
+
−
Unknown protein (AT3G12210)
BR115
+
++
+
At5g25810 mRNA
BR119
+
++
+
Unknown function
BR503
−
++
−
Unknown function
BR522
−
++
−
Hypothetical protein
BR541
−
++
−
Light harvesting complex PSII
BR547
−
++
−
UDP galactose transporter 3
+, tolerant; ++, highly tolerant; −, susceptible. Wild type, Gopum is moderately tolerant to cold but susceptible to either salinity and drought. Evaluation was based on SES for Rice (IRRI-SES, 2002)
Table 2
Composition of media used for tissue culture and transformation of rice.
Table 2
Medium
Composition
2N6 (Embryogenic callus induction)
4 g/L Chu (N6) medium (Micro and macro elements including vitamins) (Cat. No. C0204, Duchefa Biochemie B.V.), 0.3 g/L Casein hydrolysate, 0.5 g/L Glutamine, 0.5 g/L Proline, 30 g/L Sucrose, 2 mg/L 2.4-D, 2.5 g/L Gelrite (pH 5.8)
TMS membrane family protein/tumour differentially expressed (TDE) family protein
5.0
9.0
same phenotype
BR 660
translational inhibitor protein like
5.0
9.0
same phenotype
BR 670
RPL9 (RIBOSOMAL PROTEIN L9); structural constituent of ribosome (RPL9)
5.0
9.0
same phenotype
BR 700
tonoplast intrinsic protein bobTIP26-1
5.0
9.0
same phenotype
BR 708
ATPHB2 (PROHIBITIN 2) (ATPHB2)
5.0
9.0
same phenotype
BR 721
unknown protein
5.0
5.3
same phenotype
BR 725
40S ribosomal protein S4 (RPS4A)
5.0
9.0
same phenotype
BR 735
SPP2 (SUCROSE-6F-PHOSPHATE PHOSPHOHYDROLASE 2); catalytic/ magnesium ion binding/phosphatase/ sucrose-phosphatase (SPP2)
5.0
9.0
same phenotype
BR 743
mitochondrial pyruvate dehydrogenase kinase (PDK)
5.0
7.0
same phenotype
Acold stress score: 1= seedling dark green, 3=seedling light green, 5=seedling yellow, 7=seedling brown, 9=seedling dead. The cold screening was conducted at Rural Development Administration, Chuncheon, Korea
BSalinity stress score was based on the following at seedling stage: 1=Growth nearly normal, 3= Growth nearly normal but some leaves rolled sal), 5= Growth reduced and most leaves rolled(salt; only a few elongating, 7= Growth completely ceases; most leaves dry; some plants dying, 9= Almost all plants dead or dying
CObserved phenotype was based on significant differences between the wilt type Gopum using pair-wise Dunnetts multiple comparison. The same phenotype means no significant differences in the agro-morphological character such as plant height, culm length, chlorophyll contents, panicle length, number of tillers.
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Development and Identification of Transgenic Rice Lines with Abiotic Stress Tolerance by using a Full-length Overexpressor Gene Hunting System
Fig. 1
Schematic diagram of the binary Ti plasmid pFLCIII containing the full length cDNA from Chinese cabbage. pBigs vector was consisted of two different SfiI sites [SfiI(A) and SfiI(B)].
Fig. 2
PCR amplification and BLAST analyses of 933 clones. (A) Pattern and numbers of cDNA-containing fragments. (B) frequency of gene according to the length (bp) of FOX cDNA library. (C) NCBI database annotation based on gene function.
Fig. 3
Development, PCR analysis and transformation efficiency in the generation of transgenic rice. (A) Development of selected 250 clones from FL-cDNA through Agrobacterium-mediated transformation (A) callus formation; (B) inoculation; (C) callus selection using hygromycin (D), (E) and (F) shoot formation and elongation; (G) and (H) root formation and elongation; (I) acclimatization; (B) Genomic PCR confirmation of inserted genes using T1 plants by pBigs SfiI (a) and HPT (b) primers in 1% agarose gel. L= Ladder,1 = BR2, 2=BR4, 3=BR7, 4=BR23, 5=BR47, 6=BR61, 7=BR74, 8=BR91, 9=BR129, 10=BR192, 11=BR195, 12=BR174, 13=BR15, 14=BR37, WT= wild type; (C) The efficiency of Agrobacterium-mediated transformation based on the genomic PCR analysis of 1,072 T0 generation rice lines using hpt and pBigs_SfiI primers.
Fig. 4
Southern blot analysis and expression pattern of randomly selected FOX rice lines. (A) hpt gene was used as probe using a non-isotope method (dioxiginin). BR 37 (BrCIPK1, CBL-interacting protein kinase 1); BR15 (BrUGE1, UDP-glucose-4-epimerase 1); BR2 (BrCP1, Cysteine protease 1); BR4 (Actin depolymerizing factor 2); BR119 (Unknown function); BR23 (ER auxin-binding protein 2); BR29 (Phospholipase C). (B) RT-PCR of selected rice lines using their respective primer pairs (Supplemental Table 2. Rice actin was used as loading adjustments (data not shown). Gopum was the wild type.
Fig. 5
Response of FOX rice lines against cold and salinity (see materials and methods for details).
Fig. 6
Morpho-agronomic variability of 1,150 transgenic rice lines. Each transgenic rice line was compared to the wild type Gopum to generate significant differences (positive/negative) by two-pair wise Dunnet’s.
Fig. 7
The field evaluation of transgenic rice. A to C, the field plots of the transgenic lines in the vegetative stage arranged in incomplete block design; D, high senescense observed in BR 73; E, mild senescence in BR61; F, observed dwarf in BR122; G, partial sterility observed in BR 452.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Development and Identification of Transgenic Rice Lines with Abiotic Stress Tolerance by using a Full-length Overexpressor Gene Hunting System
Identified transgenic rice with cold, salinity and drought tolerance.
Clone
Tolerance to NaCl stresses (130 mM)
Tolerance to Cold
Drought (20% PEG)
Gene function (based on BLAST)
BR5
−
++
−
Unknown function
BR7
+
++
+
DEAD BOX RNA helicase RH15
BR15
−
+
+
UDP-glucose 4-epimerase
BR37
+
++
+
CBL-interacting protein kinase 1
BR39
−
++
−
Carbon-monoxide oxygenase (ACYB-1)
BR40
+
+
−
Phosphatase 2C, putative/PP2C
BR74
+
+
−
Haloacid dehydrogenase-like hydrolase
BR91
+
+
−
Unknown protein (AT3G12210)
BR115
+
++
+
At5g25810 mRNA
BR119
+
++
+
Unknown function
BR503
−
++
−
Unknown function
BR522
−
++
−
Hypothetical protein
BR541
−
++
−
Light harvesting complex PSII
BR547
−
++
−
UDP galactose transporter 3
+, tolerant; ++, highly tolerant; −, susceptible. Wild type, Gopum is moderately tolerant to cold but susceptible to either salinity and drought. Evaluation was based on SES for Rice (IRRI-SES, 2002)
Composition of media used for tissue culture and transformation of rice.
Medium
Composition
2N6 (Embryogenic callus induction)
4 g/L Chu (N6) medium (Micro and macro elements including vitamins) (Cat. No. C0204, Duchefa Biochemie B.V.), 0.3 g/L Casein hydrolysate, 0.5 g/L Glutamine, 0.5 g/L Proline, 30 g/L Sucrose, 2 mg/L 2.4-D, 2.5 g/L Gelrite (pH 5.8)
TMS membrane family protein/tumour differentially expressed (TDE) family protein
5.0
9.0
same phenotype
BR 660
translational inhibitor protein like
5.0
9.0
same phenotype
BR 670
RPL9 (RIBOSOMAL PROTEIN L9); structural constituent of ribosome (RPL9)
5.0
9.0
same phenotype
BR 700
tonoplast intrinsic protein bobTIP26-1
5.0
9.0
same phenotype
BR 708
ATPHB2 (PROHIBITIN 2) (ATPHB2)
5.0
9.0
same phenotype
BR 721
unknown protein
5.0
5.3
same phenotype
BR 725
40S ribosomal protein S4 (RPS4A)
5.0
9.0
same phenotype
BR 735
SPP2 (SUCROSE-6F-PHOSPHATE PHOSPHOHYDROLASE 2); catalytic/ magnesium ion binding/phosphatase/ sucrose-phosphatase (SPP2)
5.0
9.0
same phenotype
BR 743
mitochondrial pyruvate dehydrogenase kinase (PDK)
5.0
7.0
same phenotype
Acold stress score: 1= seedling dark green, 3=seedling light green, 5=seedling yellow, 7=seedling brown, 9=seedling dead. The cold screening was conducted at Rural Development Administration, Chuncheon, Korea
BSalinity stress score was based on the following at seedling stage: 1=Growth nearly normal, 3= Growth nearly normal but some leaves rolled sal), 5= Growth reduced and most leaves rolled(salt; only a few elongating, 7= Growth completely ceases; most leaves dry; some plants dying, 9= Almost all plants dead or dying
CObserved phenotype was based on significant differences between the wilt type Gopum using pair-wise Dunnetts multiple comparison. The same phenotype means no significant differences in the agro-morphological character such as plant height, culm length, chlorophyll contents, panicle length, number of tillers.
Table 1
Identified transgenic rice with cold, salinity and drought tolerance.
+, tolerant; ++, highly tolerant; −, susceptible. Wild type, Gopum is moderately tolerant to cold but susceptible to either salinity and drought. Evaluation was based on SES for Rice (IRRI-SES, 2002)
Table 2
Composition of media used for tissue culture and transformation of rice.
Table 3
Primers sequences used in the RT-PCR analysis and annotation of gene function by the NCBI database.
Table 4
Number of transgenic rice lines produced, confirmed gene inserts and its respective generation.
Appendix Table 1
List of transgenic rice with full-length cDNA from B. rapa, its annotation function and their morpho-agronomic traits.
cold stress score: 1= seedling dark green, 3=seedling light green, 5=seedling yellow, 7=seedling brown, 9=seedling dead. The cold screening was conducted at Rural Development Administration, Chuncheon, Korea
Salinity stress score was based on the following at seedling stage: 1=Growth nearly normal, 3= Growth nearly normal but some leaves rolled sal), 5= Growth reduced and most leaves rolled(salt; only a few elongating, 7= Growth completely ceases; most leaves dry; some plants dying, 9= Almost all plants dead or dying
Observed phenotype was based on significant differences between the wilt type Gopum using pair-wise Dunnetts multiple comparison. The same phenotype means no significant differences in the agro-morphological character such as plant height, culm length, chlorophyll contents, panicle length, number of tillers.
