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Materials and Methods
OC-I plasmids
The plasmids pLBRCys-I and pLegCys-I were already available at the University of Pretoria. These plasmids both conferred resistance to ampicillin, pLBRCys-I consisted of a cystatin (OC-I) gene under the control of a double CaMV P35S and a CaMV terminator whilepLegCys-I was under the control of a leghemoglobin promoter and a CaMV terminator (Annex 1A and B).
The presence of the transgene (OC-I) was verified by amplification of the OC-I gene using forward primer (5’-ATG TCG AGC GAC GGA GGG CC-3’) and reverse primer (5’-GAT GGG CCT TAG GCA TTT GC-3’) (Annex 2A). A 25 μL PCR mixture consisted of 12.5 μL 2X BioMix Red (Bioline, UK), 0.5 μM of each primer and 30-40 ng of template plasmid. The PCR was initiated with a 5 min hot start at 94°C followed by 30 cycles each consisting of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C. Final elongation occurred at 72°C for 5 min. The resultant PCR products were visualized on a 1% agarose gel. PCR was also carried out under the same conditions for pLegCys-I using the following primer sequences leghemoglobin forward primer (5’-CAC TCT TCA AGC CTT CTA TAT-3’) and OC-I reverse primer (5’- GAT GGG CCT TAG GCA TTT GC-3’) with annealing at 55ºC (Annex 2B). PCR products were visualized on a 1% agarose gel. The gained PCR fragments were sequenced (GATC Biotech, Germany) and verified using online tools such as NCBI blast and multalign.
Construction of plasmid LBRPRK
Phosphoribulokinase (PRK) amplification
The coding sequence of phosphoribulokinase (PRK) gene was amplified as a HindIII and BamHI (underlined sequences) flanked fragment from the template plasmid pTLT-PRK-Nter- YF (kindly provided by Norbert Mehlmer, Germany) using forward primer (5’-TCCT GGA TCC ATG GCT GTC TCA ACT ATC TAC TCA AC-3’) and reverse primer (5’-CTGT GGA TCC GCC AGA GTC AGC AGC TAG T-3’). A 50 μL PCR mixture which consistedof the plasmid (50-100 ng/μL), 5 μL 10X buffer containing 1X Mg2+ to a final concentration of 2 mM, 0.4 μL dNTPs to a final concentration of 0.2 mM, 0.25 μL of each primer (100 μM) was added, sdH2O was added to make up the mixture to 49 μL. Finally, 1 μL Pfu DNA polymerase (2.5 U) (Agilent technologies, UK) was added to the PCR mixture. The PCR cycles were 5 min at 94°C followed by 30 cycles each consisting of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C. Final elongation occurred at 72°C for 5 min. An aliquot of the PCR
product was visualized on a 1% agarose gel and an expected 200 bp band was observed.
The remainder of the PCR amplification was purified using a QIAquick PCR purification kit (Qiagen, Germany) according to the manufacturer’s instructions. Briefly, five times the volume of buffer PB was added to the PCR mix. The mixture was transferred to a QIAquick column and centrifuged. The column was washed with buffer PE. Following the wash, purified PCR product was eluted with sdH2O.
Cloning into vector pLBR
The purified PCR product and its destination vector pLBR 19 (Annex 3A) were digested with 1 U of the restriction enzymes HindIII HF™ and BamHI HF™ (New England BioLabs, USA) for 2 hrs at 37ºC in a 20 μL reaction. The digested PCR product and linearized pLBR 19 were purified using the QIAquick PCR purification kit as mentioned above (2.2.2.1).
Chapter 1: Introduction
1.1 The importance of soybean
1.2 Proteolyic enzymes
1.2.1 Classification of proteolytic enzymes
1.2.2 Cysteine protease
1.2.3 Serine proteases
1.2.3.1 Structural features of plant S8 family proteases
1.2.3.2 Attributed functions of serine proteases
1.3 Protease inhibitors
1.3.1 Cysteine protease inhibitors (cystatins).
1.3.2 Serine protease inhibitors
1.4 Plants Stress
1.4.1 What is stress and how is it perceived?
1.4.2 Drought stress and plants
1.4.3 Drought effect on plant growth
1.4.4 Drought effect on photosynthesis
1.5 Adaptation to drought.
1.6 Problem statement
Chapter 2: Construction of plasmids for transformation of soybean and Arabidopsis thaliana.
2.1 Introduction
2.2 Materials and Methods
2.3 Results.
2.4 Discussion
Chapter 3: Characterization and selection of transformed soybean plants
3.1 Introductio
3.2 Materials and Methods
3.3 Results.
3.4 Discussion.
Chapter 4: Characterization of transgenic Arabidopsis plants under well-watered and drought conditions
4.1 Introduction
4.2 Materials and Methods
4.2.1 Plant growth and maintenance
4.2.2 Plasmid and Agrobacterium strain
4.2.3 Transformation of Arabidopsis .
4.2.3.1 Generation of Agrobacterium competent cells
4.2.3.2 Agrobacterium-transformation
4.2.3.3 Generation of transgenic plants
4.2.3.4 Selection of transgenic lines
4.2.3.4.1 BASTA selection and PCR. (qRTPCR)
4.2.4 Drought tolerance analysis of transgenic plants
4.2.5 Leaf number, biomass, leaf and soil water content
4.2.6 Protein extraction, yield determination and protease activity
4.2.7 Statistical analysis
4.3 Results
4.4 Discussion
Chapter 5: Phenotypic characterization of transgenic soybean under well-watered and drought conditions
Chapter 6: General conclusions and recommendations
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SUPPLEMENTARY DATA
ANNEXURE
