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The Arabidopsis thaliana is a model organism used to study plant processes. In this paper the Eucalyptus CsID gene is investigated. Cellulose is a major component in plant cell walls. Cellulose is synthesized by cellulose synthase (CesA) genes. Agrobacterium tumefaciens is used to transform Arabidopsis plants in the floral dip method. Transformed plants are selected by antibiotic resistance using hygromycin. The GUS reporter system makes identification of transgenic plants simple. GUS staining was used for PROAtCsID3::GUS plants. PCR is used to confirm DNA extraction from transgenic plants. The chi square analysis is used discover the homozygous and hemizygous in transgenic lines.
Understanding the growth and developmental processes is made possible by studying Arabidopsis thaliana (Rhee et al. 2003). The Arabidopsis plant is a small flowering plant that has a small genome, a very short life cycle; it is easy to cross and produces lots of seeds (Naidoo 2012). These properties make the Arabidopsis an excellent plant to research the function of genes involved in a plants life cycle. Different Arabidopsis plants can differ between each other due to different ecotypes, the developmental stage or if they are mutant lines (Naidoo 2012).
There is a large amount of data on the Arabidopsis genome on an on-line resource called Arabidopsis Information Resource (TAIR), and is freely available to researchers all over the world (Rhee et al. 2003). In this program the Arabidopsis genome has been completely sequenced and regions like genes, introns and exons have been annotated. Functional genes and gene products have been analyzed, which allows researchers to obtain the genes identity, function and sequence of any gene known in Arabidopsis (Naidoo 2012).
The cellulose synthase (CsID) genes that are involved in the synthesis of cellulose have been identified using sequence analysis and expression profiles (Naidoo 2012). Cellulose is found in the cell walls of plants. There are two distinct synthesis stages of a plant cell wall (Taylor et al. 1999). The first stage is the synthesis of the primary cell wall which entails expansion by adding matrix materials and new polymers to the starting layer and then division to enlarge the cell wall. The next stage is the secondary cell wall synthesis. Compared to the primary cell wall this is a much thicker. The secondary cell wall is comprised of cellulose, hemicelluloses and lignin. Cellulose makes up 40 to 90% of the secondary cell wall (Taylor et al. 1999). The high robustness of xylem cells is due to the high proportion of lignin in the secondary cell wall (Taylor et al. 1999). Microfibrils of cellulose and hemicelluloses form the main network; lignin is then crosslinked through this network to provide the extra strength (Taylor et al. 1999). A cascade of transcription factors regulates the expression of secondary cell wall genes in a highly coordinated manor.
To transform Arabidopsis the T-DNA is transferred using Agrobacterium tumefaciens (Ditt et al. 2006). This bacterium has the ability of introducing the T-DNA into the plant cell genome using a tumor inducing (Ti) plasmid. In normal plant cells, A. tumefaciens transforms the cells into tumor forming cells by inserting part of its bacterial DNA into the plants genome. Binary vectors are used to transfer the gene of interest. In this experiment the Arabidopsis promoter AtCsID3 was inserted into the vector. This Arabidopsis promoter is the ortholog of the Eucalyptus CsID gene. The method used to transform Arabidopsis plant is floral dipping.
Floral dipping involves a solution containing Agrobacterium tumefaciens, 5% sucrose and surfactant Silwet L-77 (Clough and Bent 1998). According to Clough and Bent (1998) the highest rate of transformed progeny occurred when the plants were inoculated once many immature floral buds and a few siliques were present. The method of floral dipping is to grow the Arabidopsis to the correct stage, carefully uproot the plants, then apply the Agrobacterium to the whole plant, replant the Arabidopsis plant, collect the seeds, and finally identify the transformed progeny (Clough and Bent 1998). Antibiotic selection can be used for identification of the transformed progeny due to the presence of resistance gene in the binary vector (Clough and Bent 1998 and Naidoo 2012). The GUS gene is also present in the vector. In the construction of the vector the cloned promoter is placed right in front of the GUS gene. This will ensure the expression of the GUS reporter gene.
There are a number of ways to measure the amount of expression of reporter genes. Enzyme activity assay, immunological assay or histochemical staining of cells/tissues (Naidoo 2012). To determine if the target gene has been taken up by the cell or expressed in the cell reporter genes are used (Naidoo 2012).
In transgenic plants the GUS reporter gene enables assessment of gene activity due to the identifiable characteristics, the blue product (Naidoo 2012). The GUS reporter system is used so often due to the stability of the protein encoded, the assays are simple and sensitive and there is a wide selection of substrates available. If the reporter system is used properly there should be a correlation between the amount of expression of the reporter gene and the activity of transcription of the introduced transgenic factors (Naidoo 2012). The GUS gene encodes the β-glucuronidase enzyme. Other reporters used are Luciferase (LUC) and green fluorescent (Weigel and Glazebrook 2002). β-glucuronidase enzyme allows selection of transformed progeny to occur. The positive result of transformed progeny is a blue product; this is due to the β-glucuronidase enzyme cleaving the X-gluc synthetic substrate. This converts a colourless substrate into a blue product (Naidoo 2012).
The polymerase chain reaction (PCR) will aid in investigating the transgene. PCR is used to amplify target portions of a DNA sequence, in this case the GUS sequence. The two primers used to test for the transgene are CsIDprom_seq_1 which binds to the promoter sequence and GUS_seq_3 which binds to the GUS gene sequence (Naidoo 2012). There are another two primers used AtCsID3_promF and CsID3_prom_seq_2 (Naidoo 2012). The PCR products are then analyzed using gel electrophoresis. Gel electrophoresis is used for separating and purifying DNA fragments which can then be used for further analysis.
The aim of this experiment is use Arabidopsis thaliana plants to analyze the function of the accepted cell wall biosynthetic genes using mutant analysis, to extract genomic DNA from transgenic lines, to transfer reporter genes (eg. GUS) into the plant genome and finally to analyze crossing and segregation of PROAtCsID3::GUS plants.
Materials and Method
Growing Arabidopsis (Naidoo 2012)
The Arabidopsis thaliana plant is grown in pots, trays or peat packets (jiffies) using soil. Solid gel media can also be used as a growth substrate for Arabidopsis plants. In this experiment they were grown in jiffies. The Arabidopsis has a short life cycle, only 6 weeks. The growing conditions for these plants include a large amount of light and the ideal temperature is 22°C but temperature of 16-34°C is acceptable. Reproductive development is stimulated by long periods of light and vegetative growth is stimulated by short periods of light.
DNA extraction (Naidoo 2012)
Carefully remove 1 or 2 young leaves from the Arabidopsis plant and place these into a 1.5ml eppendorf tube. 50µl of extraction buffer is added to the tube. The extraction buffer contains 200mM Tris HCL, 25mM NaCl, 25mM NaEDTA and 0.5% SDS. The plant tissue is then grinded to break up the plant cells. Next 350µl of Extraction buffer is added, to mix properly the eppendorf tube is vortex for a few seconds. The tube is then incubated for 20 minutes at 65°C. Once this has cooled the tube is centrifuged for 5 minutes at 13000rpm. A supernatant can then been seen, and the top aqueous layer is removed and placed into a new eppendorf tube. 350µl of 2-Propanol is added to the supernatant and carefully mixed. This tube is then centrifuged for 10 minutes. The supernatant is then removed and discarded. 500µl of 70% ethanol is then used to wash the pellet. For 3 seconds the tube is then vortex and placed in the centrifuge for a final 2 minutes. The ethanol can now be removed and the pellet is air dried. 40µl of TE buffer is then added to dissolve the pellet. The TE buffer contains 10mM Tris HCL pH8.0 and 1mM EDTA. The eppendorf tube is then placed on ice.
PCR (Naidoo 2012)
First the transgene PCR is set up. 2µl of EXCEL Taq, 10µl of PCR buffer, 8µl of dNTP's, 4µl of CsIDprom_seq_1, 4 µl of GUS_seq_3, 0.4µl of DNA template and 71.6µl of distilled water are placed in an eppendorf tube. Next the second reaction (endogenous PCR) is setup. 2µl of EXCEL Taq, 10µl of PCR buffer, 8µl of dNTP's, 4µl ofAtCsID3_prom_F, 4µl of CsID3_prom_seq_2, 0.4µl of DNA template and 71.6µl of distilled water are placed into the second eppendorf tube. Positive and negative controls are setup. The negative control uses the same setup as the transgene PCR but instead of using the DNA template distilled water is used in its place. The positive control is set up like the endogenous PCR except the DNA template is replaced Wild Type Col DNA. In PCR it is important to always work on ice. These tubes are then placed in the PCR cycler where the reactions are placed under the following conditions: 4 minutes at 95°C, then 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds and 72°C for 80 seconds, which is followed by 7 minutes at 72°C and lastly 10 minutes at 4°C. The tubes are then kept at a temperature of 4°C.
Chi-square analysis (Naidoo 2012)
This is the statistical analysis of PROAtCsID3::GUS plants and is based on hygromycin resistance phenotypes. The chi square test is used to differentiate between homozygous and hemizygous. In this experiment hemizygous is when 75% of the seeds are hygromycin resistant. Seeds are collected from the parent plant and placed on medium containing the antibiotic hygromycin to determine if they are hygromycin sensitive or hygromycin resistant. This is done by first using 70% ethanol for 5 minutes to disinfect the seeds, the ethanol is removed and the seeds are placed in 10% bleach and 0.1% Triton-X solution for 15 minutes. Using distilled water the seeds are washed 3 times. Next the seeds are re-suspended in 2ml of 0.1% agar. Using plates that contain hygromycin and cefotaxime the seeds are plated out and left to dry. Once dry the plates are placed in a tissue culture room to allow germination for 5 to 10 days. One plate is used for the positive control; this contains wild type seeds and cephotaxime only in the medium. Another plate is used for the negative control; this contains wild type seeds present on medium containing cephotaxime and hygromycin. The last plate contains transgenic seeds present on medium containing cephotaxime and hygromycin. On the hygromycin plates the number of living seedlings is counted and then tested using the X² test.
X² = Σ[(O-E)²/E]
O = Observed frequency
E = Expected frequency
GUS staining (Naidoo 2012)
Two or three transgenic plants are placed into one eppendorf tube and two or three wild type plants are placed into another eppendorf tube. To make up 100ml of GUS reagent stock 29.09mg X-gluc, 16.4mg ofpotassium ferricyanide and 21.1mg of potassium ferrecyanide are added to 90ml of GUS reagent stock. 500 - 600µl of this mixed GUS reagent stock is added to each eppendorf tubes.
To get the greatest amount of vegetative growth the conditions in table 1 are recommended. The plant should be watered so that the soil is slight moist but not saturated and macronutrients should be supplied once a week, this can be in the form of commercial fertilizers (Weigel and Glazebrook 2002).
Table 1. Conditions for Arabidopsis growth (Weigel and Glazebrook 2002).
Cool fluorescent bulbs or shaded sunlight
Less than 12 hours of day light
DNA was successfully isolated from positive plants.
To confirm the transgenic plant PCR was done. The PCR products are analyzed using gel electrophoresis. The results of electrophoresis are in figure 1. The molecular marker is in lane one. Lane two and lane three is the trangene. Lane four and five is the endogenous gene. Lane six is the positive control and lane seven is the negative control. The expected size of the transgene is larger than the endogenous gene.
Figure 1. The gel electrophoresis products comparing transgenes and endogenous genes.
The null hypothesis: There will be 100% homozygous Arabidopsis plants for the transgene for hygromycin resistance by T6 and T7. The number of observations in each category is equal to that predicted by a biological theory.
The alternative hypothesis: There will be hemizygote/heterozygote Arabidopsis plants for the transgene that confers hygromycin resistance in the T6 and T7 generation. The observed numbers will be different from the expected numbers or ratio.
X² = 7.314
7.314 > 6.635 at 99% significance
7.314 > 3.841 at 95% significance
Thus the null hypothesis is rejected.
Table 2.Tthe results of GUS staining
The plant is a white colour
The plant is a blue colour
Arabidopsis thaliana has a short life cycle of 6 weeks. If the plant is under conditions of long periods of light this will stimulate reproductive development and the life cycle will be even short. If the light periods are short vegetative growth is stimulated.
The floral dip method is an easy way of transforming Arabidopsis plants using Agrobacterium. Some problems associated with floral dip are that in order for the method to work properly only a very specific ecotype must be used and double transformation may occur which will inevitably lead to a false homozygous result after segregation.
Homozygous is when the same allele is present at one locus on both chromosomes. Heterozygous is when different alleles are present at one locus on each chromosome. Hemizygous is when a chromosome has an extra piece of DNA therefore when you line up the two pieces there is a homolog lacking which forms a bubble. These are important when introducing genes into desired genotypes.
When using the chi square analysis, the test will be more accurate if lots of seeds are analyzed. Seed clumping makes analysising the seedling phenotypes difficult which could the final results. The transgenic plant lines growing in the hygromycin saturated agar can be identified by their root system embedded in the agar. The positive control used has wild type seedlings and no antibiotic present in the agar thus all plants will grow. This control is used to make sure the plant are viable and can grow under these conditions. The negative control used has wild type seedlings present in hygromycin saturated agar thus no plants will grow. The wild type plants do not have the hygromycin gene and are thus sensitive to hygromycin.
Once the genomic DNA is extracted from the transgenic lines PCR is done to check the insert of interest is present.In the PCR reaction there are a number of controls used. The positive control definitely contains the trangene. This is used to make sure the reaction works, this result is what you except to see if the trangene is present in the target gene. There are two negative controls, the first contains water. This control contains the primers but not the DNA template. This is used to test for contamination. The second negative control contains the transgene but the temple used does not contain the transgene. This is used to test whether the target gene is the correct region. The PCR products are analyzed using gel electrophoresis. The positive control used here is the wild type gene in bacterial Escherichia coli. The negative control is distilled water, used to test for contamination.
GUS staining is used to analyze gene expression. The wild type plant turns white this is due to the wild type plant not having the GUS reporter gene thus it won't be able to break down X-gluc. Whereas the transgene has the GUS reporter gene therefore can break it down, turning the substrate blue. The negative control used contains the wild type plant. This result confirms that the reporter is directly attached to the target gene which creates a gene fusion.
All these techniques used have impacted the amount know about plants and plant processes. These techniques can be used for future research into food security, genetically modified organisms, crop protection and improved growth of plant crops.