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Critiquing the Analysis on the Enhancement on Iron and Zinc Accumulation in Genetically Engineered Wheat Plants through Overexpression of Sickle Alfalfa ferritin
The common wheat, Triticum aestivum, belongs to the genus Triticum, and the family Graminae, or, the grass family (Canadian Food Inspection Agency, 2014). The common wheat is described to be a mid-tall annual grass consisting of flat leaf blades with a terminal floral spike (CFIA, 2014).
This spike consists of perfect flowers (male and female reproductive systems in the same flower). Culms (or stems of the plant) are comprised of five nodes with four foliage leaves, one of which, the flag leaf, subtends the inflorescence. The inflorescence consists of seed-producing flowers that contain specialised branches called spikelets (Dixon et.al., 2018). The spikelets consists of two glumes that enclose approximately nine florets and are then alternately arranged on the rachis (CFIA, 2017). Figure 1 below represents the various components of the wheat plant in detail.
T.aestivum is a hexaploid (so: AABBDD), an organism containing six sets of chromosomes, 42 in total (Australian Government, 2008). Various other wheat species contain a form of haploid with a set of seven chromosomes (CFIA, 2014).
Tetraploid (i.e. durum wheat, AABB) and hexaploid are the two modern wheat cultivars used.
T.aestivum – common wheat – is often regarded as the most widely used and domesticated food crop in the world (Courteau, 2018). It is also the most commonly used in all of the cultivated wheats. Approximately 20% of food calories and protein nutrition are due to wheat consumption, and, a further 40% of the world’s population consume wheat (Gupta, 2008).
Figure 1: Various Components of The Common Wheat Plant
Iron is one of the most vital dietary foods required for a variety of bodily functions (Better Health, 2018). Iron deficiency is a common health issue, and, according to the World Health Organisation, approximately 2 billion people are affected by (iron deficiency) anaemia globally (WHO, 2005). A further four to five billion people suffer from iron deficiency, with symptoms including fatigue, headaches, dizziness and shortness of breath (Healthline, 2017). Fortification of common wheat may be a potential solution to assist in the alleviation of iron deficiency, mainly as it is cultivated all over the world (40% of the population) (Liu et.al., 2015).
The report focusses on the over-expression of the ferritin gene – located in Medicago falcata L, sickle alfalfa – in order to increase iron and zinc concentrations in transgenic crops (Liu et.al., 2015). The ferritin gene is vital in iron accumulation regulation in plants and already proven effective for increasing iron levels (Kanobe et.al., 2013).
Before Genetic Transformation: The Isolation of ferritin from sickle alfalfa
Before any transformation was undertaken, isolation of ferritin from sickle alfalfa occurred first. Harbin Normal University in China was where the sickle alfalfa were grown, and, as the seedlings reached maturity with three leaves, RNA extraction was conducted on the leaf samples (Liu et.al., 2016). Total RNA was extracted in order to prepare for the latter stages of the method, where reverse transcriptase PCR (RT-PCR) was performed. RT-PCR involves the extraction of either total RNA or messenger RNA (mRNA) where it is then transcribed into complementary DNA (cDNA, however more to come) (ThermoFisher, 2018).
Plant material that was genetically transformed
Wheat cultivar: Longfu16
Immature embryos (13-14 days postathesis) were the explant, and used potentially due to its efficiency in regenerating whole plants in abundance (ResearchGate, 2014). The immature embryos were cultured in callus, in an induction medium consisting of Murashige and Skoog (MS) media, 2, 4-dichlorophenoxyacetic acid (2mg/L), 50g/L of sucrose and 5g/L of agar. 2, 4-dichlorophenoxyacetic acid is a synthetic auxin, which promotes cell division and growth (plant growth regulator). When applied to dicots, 2, 4-D is known to kill them due to uncontrollable and unsustainable growth within the plant tissues, however, has no effect on monocots, which includes T.aestivum (Clark and Pazdernik, 2016). Its use was in moderation, with 2mg/L, and, along with the other components within the medium and the explant, cultured in the dark for 7 days, a period of time said to be sufficient in regards to callus development (Dagustu, 2014).
Sucrose, a carbon source incorporated in the media, is essentially the energy source for the immature embryos and the MS media, or gelling agent, provided solid support.
The culture conditions for the explant/induction media was to culture the explants for 7 days in the dark, at a temperature of 25oC, the ideal conditions for in vitro callus growth and induction (Kumar, 2018).
‘nos’ – Nopaline synthase – was the terminator in the vector. The termination sequence from this gene was originally isolated from Agrobacterium tumefaciens, a Gram-negative bacterium (Collins, 2001). The function of the nos sequence is to essentially stop transcription (expression) of the ferritin gene (European Commission, 2009).
The GluB-1 gene was the expression promoter for the sickle alfalfa ferritin gene. GluB-1 promoter is a seed-storage protein controlling the expression of ferritin. A second promoter, ‘35s’, represented the cauliflower mosaic virus (CaMV) promoter. The selectable marker was the bar gene. See Figure 2—the expression vector (pBlu-GluB-1/Fer).
Figure 2 is the diagram of the plant expressing vector (pBlu-GluB-1/Fer) used for plant transformation. Nos-terminator; Ferritin-Gene of Interest; GluB-1-expression promoter; 35s-Cauliflower mosaic virus derived promoter (CaMV 35s); bar—selectable marker; first two arrows indicate restriction sites. HindIII, BamHI, SacI and bg1II are the restriction enzymes.
The 35S promoter had originated from the cauliflower mosaic virus, and results in high gene expression levels in dicot plants and monocots (Furtado et.al., 2008). CaMV is constitutive, and can be used in the majority of tissues, however, because of this, unnecessary effects in transgenic plants (for instance pleiotropic) are likely to occur in transgenic plants (Furtado et.al., 2008). The authors used GluB-1, a seed-specific gene promoter, originating from rice seeds (Sarker et.al., 2015; Furtado et.al., 2008). The GluB-1 promoter was most likely used as it was shown to increase iron concentrations in transgenic T1 rice; the soybean ferritin gene was expressed in a separate experiment, in the transgenic T1 rice (Goto et.al., 1999). Ubiquitous promoters, for instance those found in wheat, are unstable, often ineffective in the expression of recombinant protein genes (Furtado et.al., 2008).
Present in animals, bacteria and plants, ferritin is a fundamental iron storage protein located within the cells (intracellular) (Weizmann Institute of Science, 2018). Vital in iron homeostasis, ferritin is also involved in the delivery of iron to the cells in the body, further providing iron through the synthesis of ferredoxin and cytochrome (Weizmann Institute of Science, 2018; Goto et.al., 1999). Both are examples of iron proteins. These may be potential reasons as to why ferritin was used in the experiment, as it is directly involved in transporting iron in the cells, as well as for storage. The overexpression of ferritin may lead to an increase in iron levels in transgenic wheat plants, which may further assist in reducing levels of iron deficiency around the world.
HindII, BamHI, SacI and Bg1II are restriction enzymes, all of which have an optimum temperature of 37oC (Thermo Fisher, 2018). HindIII will recognise and cut sites that are present with A^AGCTT (Thermo Fisher, 2018).
BamHI is a type II restriction endonuclease derived from the bacterium Bacillus amyloliquefaciens (Sigma-Aldrich, 2018). BG1II will cut A^GATCT sites (Thermo Fisher, 2018).
SacI will cut sites that have the following sequence: GAGCT^C.
After the 7-day duration ended, the target tissue (unspecified, though explants/cultures, not quite embryogenic callus) was transferred to a medium that had contained 0.2M mannitol for 4-6 hours prior to particle bombardment (and 16-18 hours after bombardment). Exposing the target tissue to mannitol prior to particle bombardment would have allowed for osmotic treatment enhancing the process (of particle bombardment), potentially reducing cell damage (Vain et.al., 1993). Specifically, the extrusion of the protoplasm (living components within a cell) from the bombarded cells would have been prevented (Vain et.al., 1993). Gold particles coated with the expression vector (pBlu-GluB-1/Fer) were used for particle bombardment. Gold beads are preferred over tungsten beads due to tungsten beads being potentially toxic to several plants (Clark, Pazdernik and Pazdernik, 2010). Specific information in regards to the type of gun, pressure or distance were not discussed, however Figures 3 illustrate two types of particle guns for DNA (in this case, cDNA).
Figure 3: A) An example of a gene gun that utilises pressurised air; B) a second example, however, instead of pressurised air, the gun utilises a high-voltage discharge.
After bombardment the cultures were transferred to callus induction medium and incubated for 14 days in the dark before being transferred to selection. This was to ensure and allow for thorough differentiation with regenerative/non-regenerative shoots once placed in bialaphos (from the bar gene, selectable marker).
The bar gene was the selection marker in the experiment, and is essential for producing herbicide resistant plants as it is resistant to bialaphos (herbicide) (Patent Lens, 2018). The embryogenic callus was transferred to a ½ MS medium along with 5mg/L zeatin and 2mg/L bialaphos – the primary selection agent – and maintained under light (Liu et.al., 2016). At a temperature of 25oC, the plants were regenerated due to the presence of bialaphos. Bialaphos is an effective selection agent, and, along with zeatin, promotes efficient regeneration in plants (Aftabi et.al., 2018). There were resistant regenerated shoots: using ½ MS (so half strength) medium with 0.3mg/L IAA and 0.5mg/L MET for 14 days ensured cell growth and division in the shoots. They were then placed in pots in a greenhouse at a temperature of 28oC, with a luminous emittance of 25, 000 (i.e. full daylight, not from sun). Specific conditions were also made with exposure to light/dark: 16/8 hours (16 hours light, 8 hours dark).
Characterisation of the Transgenic Plants
The first technique used to characterise the transgenic plants was standard PCR. This was used to determine the presence of the sickle alfalfa ferritin in the transgenic wheat and positive control. The primary PCR condition was a temperature of 94oC for 5 minutes, in order to denature the DNA strands, exposing the nucleotide bases (Khan Academy, 2018). The use of Taq polymerase enzyme synthesised two brand new strands of DNA with one strand being the original, the other ‘new.’ (National Human Genome Research Institute, 2016). There were 35 cycles in total. Genomic DNA was extracted from the transgenic plants (T1), with the non-transformed wheat plant representing the negative control, the plasmid DNA the positive control. Along with Taq polymerase buffer being added into the PCR solution mixture, dNTP was also added. DNTP is a nucleotide phosphate, used in PCR amplification (Babec, 2012). Fer-1 and fer-2 were the two primers incorporated in the mixture. Taq polymerase would not have been able to make DNA without fer-1 and fer-2. In this experiment, they would have been created such that they were ‘aimed’ at the target region, providing the necessary sequences enabling them to bind to the opposite strands of the original DNA template (Khan Academy, 2018).
Figure 4 below was the result of the standard PCR analysis after being analysed by 1% agarose gel electrophoresis. 500bp was correspondent to the expected size of ferritin, and was found to be present only in the transgenic wheat and positive control (as it was absent in the non-transformed wheat which was the desired outcome) (Liu et.al., 2016).
Figure 4: Standard PCR analysis result. At a length of 500bp the sickle alfalfa ferritin was present in the transgenic wheat and the positive control. It was not evident on the band representing the non-transformed wheat. P:positive control, NT: non-tranformant
Molecular Analysis through Reverse Transcriptase PCR
Reverse transcriptase involves synthesising DNA from an RNA template and producing complementary DNA, or cDNA (New England BioLabs, 2018). Using RTs is effective and ensures that the completion of the mRNA transcript at the 5’ end has been successfully copied (New England BioLabs, 2018).
The experiment used PCR-positive wheat plants for RT-PCR in order to ascertain the transgene expression. The total RNA was isolated from both the T1 transgenic wheat and the non-transformed wheat plants with 1mg total RNA incorporated for the first strand cDNA synthesis. 5ml of the cDNA solution was provided for the template and fer-1 and fer-2 primers were again used for PCR amplification. The set up was similar again with the plasmid DNA was the positive control, and the temperatures and cycles the same as above PCR analysis.
Figure 5 is the result of the RT-PCR analysis after electrophoresing. The RT-PCR analysis had produced a 500bp band, as did the PCR above. The 500bp used the total RNA from the transgenic wheat as well as the positive control only. Another successful result, this meant that the total RNA from the non-transformed wheat plant was not evident, meaning that the ferritin had successfully integrated into the transgenic wheat.
Figure 5: Analysis of Transgenic Wheat after using RT-PCR. The 500bp indicated that the total RNA from the transgenic wheat and the positive control was used. This further meant that the sickle alfalfa ferritin had been incorporated in the transgenic wheat. P:positive control; NT: non-transformant
Antibody Detection of Proteins Using Western Blot Analysis
Western blot analysis detects and analyses proteins (Human Protein Atlas, 2018). This was used in order to inspect the expression levels of the sickle alfalfa ferritin in the wheat. The paper had used three T1 transgenic plants and one non-transformed wheat plant. Seeds from the plants were used in order to extract the total proteins. These were then ground into powder with the following, all of which were buffer preparation for the western blot analysis: 200 mM Tris-HCl pH8.0, 100 mM NaCl, 400 mM sucrose, 10 mM EDTA (ethylenediaminetetraacetic acid), 14 mM 2-mercaptaethanol and 0.05% (w/v) Tween-20. Tris buffer is essential for protein analysis, particularly western blotting and protein electrophoresis and helps maintain the pH (BioRad, 2018).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used for the western blot analysis; the polyacrylamide gel was used in the electrophoresis as a supporting medium, whilst the sodium dodecyl sulfate denatured the proteins (Caprette, R.D., 2012).
Figure 6 was the result of the western blotting. The bands on lanes 1-3 had a 28KDa band on them, which was indicative of the presence of transgenic wheat grain protein. The transgenic protein was not detected with the non-transformed wheat grain: no contamination within the non-transformed wheat grain band as a result of this successful result. This was another result in which the sickle alfalfa ferritin was successfully expressed in the transgenic wheat grains.
Figure 6: Western Blot Analysis of ferritin. P—positive control; NT: non-transformant
Determining Thousand Grain Weight and Measuring Mineral Concentrations
The last experiments conducted were thousand grain weight (TGW) calculations and determining the grain mineral concentrations in the transgenic and non-transformed wheat plants. Data from both procedures were to be used in an analysis for variance (ANOVA) to ascertain if there was any significant difference between the transgenic and non-transformed wheat plants. This would have further supported the analyses above, allowing for the researchers to determine how much of the minerals – and which type (e.g. Iron/Fe, Zinc/Zn) – were increasing/decreasing. Thousand grain weight refers to wheat seed weight, a factor known to contribute to an increase in wheat yields over the past decade and a factor influencing grain quality (Feng et.al., 2018).
Nutrients such as potassium sulfate (K2SO4), ammonium phosphate dibasic [(NH4)2HPO4] and urea [CO(NH2)2] were mixed into soil with pots containing black and marsh soil. In order to prevent drought stress, the plants were subjected to irrigations in their cycle. The grains of transgenic wheat – G3 – and non-transformed wheat were used for TGW, with a total of 250 grains used. The estimates were averaged out three times. Inductively coupled plasma-atomic emission spectrometry was the technique used to calculate the mineral concentrations, as it is known to be cost and time efficient, determining trace and major elements in minerals in under two minutes (Jarvis and Jarvis, 1992). Zinc, Iron, Calcium, copper, cadmium and manganese were the minerals used for concentrations.
The table in Figure 7 outlines the significance values for each mineral and TGW value correspondent to the transgenic wheat lines (P1 – P4) and non-transformed (NT). The prominent finding in the results was that the iron (Fe) and zinc (Zn) concentrations in the transgenic wheat lines had increased by 73% and 44% when compared to the non-transformed plants.
Figure 7: Analysis on The Seeds of the Transgenic Wheat and Non-transformed. NT: non-transformed; P1-P4 represent the transgenic wheat seeds. The upper case letters signify a significant difference in the appropriate element and/or Thousand Grain Weight (TGW) when p<0.01. The transgenic lines in P1 and P2 were significantly higher than those for the non-transformed wheat, p<0.05. The grain concentration for Fe and Zn in the transgenic lines P1-P4 had increased significantly in comparison with the NT.
It was evident in the molecular analyses that the overexpression of the sickle alfalfa ferritin had caused an increase in iron concentration in transgenic wheat plants by 73%. The paper had also aimed to potentially assist in the reduction of iron deficiency with the uses of ferritin in wheat plants, however, genetically engineered crops have been a controversial talking point in regards to food labelling and the environment.
GM/GE food labelling is a difficult issue surrounding consumer rights and their choice on genetically engineered crops (whether they wish to consume genetically engineered wheat) (Maghari and Ardekani, 2011). This may affect further purchase of the relevant GM product (in this case, wheat plants with ferritin).
In countries such as the US mandatory labelling is required, however, on a global scale, it is still unclear – and unlikely – that a labelling system will be implemented (Maghari and Ardekani, 2011). Already used in more than 40 countries, research has shown that people around the world sought for transparency and consumer choice, further believing that the compulsory labelling on GM ingredients be required (Maghari and Ardekani, 2011). Those that oppose GE/GM crops argued that such labelling would repel consumers from purchasing a product that has been genetically engineered (Maghari and Ardekani, 2011). A study in America showed that, whilst 93% of people believed that the government should incorporate ‘GM’ labelling in foods, 58% of them further claimed they would not purchase any product with such labelling (Ethical GMO, 2011). Even if there would be an attempt to sell wheat plants with ferritin, there is no certainty that it would sell due to the government/s seeking to meet the demands of the consumers (Ethical GMO, 2011). This would affect the business’ sales of the product, struggling with potential extra costs to pay for lack of GE/GM sold (Ethical GMO, 2011).
Producing more crops with genetically engineered wheat plants seeking to overexpress ferritin may be difficult as there are potential environmental risks associated with GM crops. Cross pollination in particular: when the cross breeding between a genetically engineered crop and a non-GM/GE crop occurs (Polya, 2001). Cross pollination has the potential to result in changes and incursions within the gene pool and genome of ecosystems, ‘natural’ species (Polya, 2001). Should transgenic wheat be planted in a field, buffer zones may have to be implemented to ensure cross pollination does not occur.
Word Count: 2867 Excluding Titles, Figure Details and References. (page 9-current)
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