Expression of oryzacystatin-i in drought and heat-stressed transformed tobacco plants

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Detection of OC-I sequence in transformed plants 

Genomic DNA was isolated from putative young leaves of transformed and control tobacco plants using a commercial DNA isolation kit (Amersham Phyto Pure DNA isolation kit, Amersham, UK). The quantity and quality of genomic DNA was determined by the NanoDropÒ reading technique. A standard Polymerase Chain Reaction (PCR) using 100 ng genomic DNA as template was applied to amplify a portion of the OC-I coding sequence from transformed tobacco plants with a OC-I forward (5’- TCACCGAGCACAACAAGA-3’) and reverse (5’– CATCGACAGGCTTGAACT – 3’) primer. Plant DNAs, from which DNA bands of the expected size of 200 base pairs (bp) could be amplified and visualized on a 1.5% agarose gel, were considered as transformed with OC-I. These plants were labelled and transplanted to 5 l capacity pots containing 1:1 river sand/coconut coir potting media along with a corresponding non-transformed control plant and used for induction of drought, heat and combination of drought and heat stress two weeks after transplanting.

Stress treatment of plants

The experiment was conducted at growth cabinate facility situated on experimental farm of the University of Pretoria, South Africa. The two types of tobacco (Samsun) plants, transformed and non-transformed (non-transformed plants used in all experiments described in this thesis are obtained from segregating population of primary transformants after twice selfing) were treated with two growth temperatures consisting of treatments at: 26/20 ± 2°C (normal temperature) and 38/30 ± 2°C day/night (heat treatment) at 12 hours light photoperiod, and also two water regimes consisting of treatments with 25 – 35% (drought stress) and 80 – 100% field capacity (non-drought stress). This experimental design resulted in a 2 x 2 x 2 factorial treatment combination in a randomised complete block design, where each treatment set was replicated ten-times.
Drought stress treatment was induced based on a gravimetric method. Watering was done on a daily basis by weighing individual pots based on the field capacity determination and the treatments. Light in the growth cabinet was provided by a combination of incandescent and fluorescent lamps generating a photosynthetic photon flux density of 240 ± 10 mmol m–2 s-1. The relative humidity in the individual cabinet during the study period was 60 ± 4%. Plants received Hoagland nutrient solution three-times a week. Four weeks after treatment induction, leaf samples were collected from fully expanded 3rd or 4th leaf position from shoot tip, flash frozen in liquid nitrogen and either immediately used or stored at -80°C until needed. The entire experiment was repeated twice. Leaf samples from both experiments were used as DNA and RNA sources in the analysis.

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Preparation of leaf protein extract

Frozen leaf samples were homogenized in liquid nitrogen in the presence of an extraction buffer. The buffer contained 50 mM Tris-HCl, (pH 8), and to block proteinase activity, 1 mM PMSF (phenylmethylsulphonyl fluoride), 1 mM EDTA (Ethylenediaminetetraacetic acid), 10 μM trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64) and 10 μM pepstatin A. The homogenate was centrifuged at 13000 rpm at 4°C for ten minutes in an Eppendorf centrifuge (Eppendorf, Gemany) and the resulting supernatant was used for further analysis after determination of the protein concentration according to Bradford (1976) using BSA as a standard (Bio-Rad, Hercules, USA).

CHAPTER ONE
1.1 Plants and stress
1.2 Plant gene expression under stress
1.3 Plant engineering for drought and heat stress tolerance
1.4 Proteinase/proteinase inhibitor system and stress
CHAPTER TWO
EXPRESSION OF ORYZACYSTATIN-I IN DROUGHT AND HEAT-STRESSED TRANSFORMED TOBACCO PLANTS
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.4 Results
2.5 Discussion
CHAPTER THREE
GROWTH OF OC-I EXPRESSING TRANSFORMED TOBACCO PLANTS UNDER ABIOTIC STRESS
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.4 Results
3.5 Discussion
CHAPTER FOUR
APPLICATION OF cDNA REPRESENTATIONAL DIFFERENCE ANALYSIS (cDNA RDA) FOR DETECTION OF DIFFERENTIALLY EXPRESSED GENES IN OC-I EXPRESSING TOBACCO
4.1 Abstract
4.2 Introduction
4.3 Materials and Methods
4.4 Results
4.5 Discussion
CHAPTER FIVE
CLONING OF TWO NEW CYSTEINE PROTEINASES WITH SPECIFIC EXPRESSION PATTERNS IN MATURE AND SENESCENT TOBACCO LEAVES
5. 1 Abstract
5.2 Introduction
5.3 Materials and methods
5.4 Results
5.5 Discussion
CHAPTER SIX
SUMMARY AND PERSPICTIVE
REFERENCES

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