Developing Drought Tolerance In Sunflower Biology Essay

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Developing drought tolerance in sunflower through a focused trait based breeding has been receiving great emphasis in recent years. Success of such an effort depends on the identification of promising traits donor lines and their validation under water limited conditions. From this context, assessing the genetic variability in relevant traits such as root traits and WUE assumes importance.

The methodology adopted for screening a diverse set of sunflower genotypes for variability in roots and WUE is described in this chapter.

The transcription factors (TFs) acts as key regulators of functional genes associated with abiotic stress responses. Therefore, engineering of TFs genes can be attempted to induce the coordinated expression of functional genes involved in stress tolerance. There are many reports on over expression of a single TF giving tolerance to multiple stresses.

Plant material

Sunflower genotype IB20 was used for transformation study which was procured from Sunflower Scheme, University of Agricultural Sciences, Bangalore, India.

Bacterial strains and vectors

The Agrobacterium strain EHA 105, harbouring the binary vector pGreen 0179 (pG-CaMV 35S-EcNAC1 casset) (Figure 1) was used for transformation of EcNAC1 transcription factor gene which was procured from the Department of Crop Physiology, University of Agricultural Sciences, Bangalore, India. In this binary vector hygromycin phosphotransferase (hptII), 35S CaMV, nos gene are the marker, promoter and terminator sites respectively.

Development of transgenic sunflower plants

Agrobacterium- mediated in planta transformation of Sunflower

A single colony of Agrobacterium harboring recombinant binary vector was grown in LB medium containing 30 µg/ml hygromycin overnight at 28C. The bacterial cells were later resuspended in Winan's AB medium (pH 5.2) and grown for 18 hours. Wounded tobacco leaf extract was later added to this suspension and kept ready for infection.

Plant material Preparation

Total 100 seeds of Sunflower genotypes IB20 were soaked in water over night and later surface sterilized with 0.1% mercuric chloride for 5-7 minutes, followed by thorough rinses with sterile distilled water and allowed to germinate on Petri plates for two days. The germinated seeds are ready to use for Agrobacterium infection.

Agrobacterium infection and recovery of transformants

The infection was carried out by pricking randomly on the actively dividing region with a 28 gauge-sewing needle. Wounded seeds were infected with Agrobacterium, cultured in the AB minimal medium and kept for gentle agitation at 28ï‚°-30ï‚°C, for 45 minutes and then the seedlings were blot-dried and washed thoroughly with distilled water and placed on autoclaved soil rite for germination under aseptic conditions in capped bottles. About 70 percent of the seeds germinated under aseptic condition. After 5-6 days, the germinated seedlings were transferred to soil rite in pots and the seedlings were allowed to grow under growth room conditions for at least 10 days and later they were transferred to green house. Under green house condition plants showed normal growth and development.

Selfing and collection of seeds from T0 sunflower

Sunflower is an extremely cross-pollinated crop, to avoid pollen contamination, T0 plants were covered with cloth bag after initiation of flower. Self pollination of T0 plants were done by rubbing flower head with cloth bag twice a day for a period of 10 days. After pollination, cloth bags were removed from the head for ensuring proper aeration. After maturity, heads from survived plants (24 numbers) were removed and allowed for sun drying. T1 Seeds from the dried heads were dehusked and used for analysis.

Screening Putative Sunflower Transformants

The preliminary screening of putative sunflower transformants was carried out using salt (NaCl) stress method. The salt tolerant plants were selected for further molecular analysis.

Standardization of stress inducers concentration

Leaf discs from fully expanded leaves of 6-8 weeks old wild type sunflower (IB20) plants were subjected to 50, 100, 150, 200 and 250 mM NaCl concentration, incubated for 48 hr at room temperature. The induction of chlorosis in the leaf discs were recorded after 48 hr incubation. The final screening concentration was fixed based on the concentration at which the green colour leaf discs turns to complete dark brown or black.

Screening putative transformants by salt stress induced leaf damage

Leaf discs from fully expanded leaves of 6-8 weeks old putative transformants and wild type were placed on filter paper dampened with 200 mM NaCl. They were incubated for 48 hr at room temperature and the symptom of bleaching or browning was documented. The salt tolerant plants were selected for further molecular analysis.

Standardization of Ethrel concentration

Leaf discs from fully expanded leaves of 6-8 weeks old wild type sunflower (IB20) plants were subjected to 250, 500, 750, 1000 and 1250 ppm ethrel concentration, incubated for 48 hr at room temperature. The induction of chlorosis in the leaf discs were recorded after 48 hr incubation. The final screening concentration was fixed based on the concentration at which the green colour leaf discs turns to complete dark brown or black.

Response of putative transformants under ethrel (1000 ppm) stress

Leaf discs from fully expanded leaves of 6-8 weeks old putative transformants and wild type were placed on filter paper dampened with 1000 ppm ethrel. They were incubated for 48 hr at room temperature and the symptom of bleaching or browning was documented.

Standardization of methyl viologen concentration

Leaf discs from fully expanded leaves of 6-8 weeks old wild type sunflower (IB20) plants were floated on 2, 4, 6, 8 and 10 µM methyl viologen concentration and incubated for 5 hr in direct sunlight. The induction of chlorosis in the leaf discs were recorded after 5 hr incubation. The final screening concentration was fixed based on the concentration at which the green colour leaf discs turns to complete dark brown or black.

Response of EcNAC1 transgenics under methyl viologen (4 µM) stress

Leaf discs from fully expanded leaves of 6-8 weeks old putative transformants and wild type were floated on 4 µM methyl viologen solution. They were incubated for 5 hr in direct sunlight and the symptom of bleaching or browning was documented. Further the leaf discs were used to estimate the chlorophyll content and cell membrane stability.

Estimation of total chlorophyll content

Total 100 mg of leaf tissue from control plants (wild type and EcNAC1 transgenic lines) were suspended in test tube containing 10 ml of acetone (80%): DMSO (1:1 mix) solution and allowed to bleach completely. After complete bleach, the absorbance was recorded at 663, 652 and 645 nm using UV-visible spectrophotometer (UV 2450, Shimadzu Corporation, Kyoto, Japan). Total chlorophyll content was estimated by following the methods described in Hiscox and Israelstam (1979). The chlorophyll content was also estimated from the same plants after salt and methyl viologen stress imposition. The calculations are given below

Total chlorophyll (mg/g FW) = chlorophyll a + chlorophyll b

Estimation of cell membrane stability (CMS)

Leaf discs from wild type plants were initially rinsed to remove solutes leaking at cut end surface and incubated in deionised water for overnight (12 hr) at 25oC. The extract of electrolyte that leaked into bathing medium was recorded (T1) using conductivity bridge (Elico CM183 EC-TDS analyzer, Bangalore, India). Subsequently, the leaf discs were incubated in 65oC for 1 hr and allowed to cool down and final reading was recorded (T2). Similarly leakage was also measured from EcNAC1 transgenic lines were incubated with 200 mM NaCl solution for 48 hr and methyl viologen (4 µM) for 5 hr. After incubation period the leaf discs collected from the same set of plants after salt and methyl Viologen stress imposition (C1 and C2). The membrane stability was calculated using the formula: CMS (%) = [1-(T1/T2)] / [1-(C1/C2)] x 100.

Observations recorded during the crop growth stage.

Following observations were recorded in each genotype with two replications using plants raised in cement root structures. In each line four plants were labelled for taking observations. At vegetative stage

Plant height (cm/pt):

Height of the plant was recorded from base of the stem to the base of the capitulum at flowering.

Number of leaves/plant:

Numbers of leaves were counted from top of the plant towards base.

Total leaf area (TLA):

Total leaf area, an indication of total photosynthesizing area of the plant was measured following non destructive method proposed for sunflower by Nanja Reddy et al., (1995). According to this method, leaf area was determined by counting the total number of leaves initially and multiplied it with a constant value 0.369 to arrive at the position of the leaf from the top to be considered for length (cm) and width (cm) measurement. If this value is turned out to be a fraction, then the value was rounded off to the nearest number. Once the length and width were measured, value obtained was again multiplied with a constant value 0.69 and the total number of leaves to arrive at the total leaf area, which was expressed in cm2/pt.

Total leaf area (cm2/pt) =length x breadth of index leaf x 0.369 x total number of leaves per plant.

Specific Leaf area:

It is an indication of leaf area per unit leaf weight. The fully expanded leaf more specifically, fifth leaf from the top was used for SLA measurement. After measuring the maximum length and breadth of the leaf, the leaf was kept in a oven at 80á´¼C for 3 to 4 days, the dry weight (g/pt) was recorded. Then the specific leaf area (SLA) was calculated by using the following formula.

Total leaf area

SLA (cm2/g) =

Leaf dry weight

∆13C (‰):

A small leaf sample was collected from the field and dried overnight at 80ᴼC, powdered & used for ∆13C estimation by Isotope Ratio Mass Spectrometer (IRMS). Fifth leaf from the top which was used for SLA measurement was taken for carbon isotope discrimination studies. After complete drying of the leaf, the leaf was grinded and made into fine powder. While grinding, care was taken to see that there is no contamination of the sample by washing the ball mill every time with acetone. Approximately, 1mg of fine powdered sample was taken for 13C analysis using Isotope Ratio Mass Spectrometer.

Stem dry weight (g/plant):

Stems were dried at 80°C, and then dry weight was recorded for individual

plants.

Root dry weight (g/plant):

The roots were put in the kraft paper bags and dried at 80á´¼C. Dry root weight was measured on the digital balance.

Root length (cm/pt):

Root length was measured using long scale from top of the root system to its tip immediately after the harvest.

Root volume (cc/pt):

Root volume was measured by water displacement method. Known amount of water was taken in a measuring cylinder, root was immersed completely in water and the increase in water level was recorded as root volume in cc.

Harvesting:

The crop was harvested when it attained full maturity. Leaves, stem and thalamus were separated from the plant and dried in the oven at 80á´¼C and dry weight was recorded when constant weight was attained.

All the parameters recorded in root structure were also measured in field except root traits as explained in 3.1. In addition the following parameters were also recorded.

Stem diameter (cm/pt): The diameter of the stem between the 6th and 7th true leaves was measured using vernus calipers.

Grain yield:

Seeds were separated from the head, dried and weighed and expressed as gram per plant.

Harvest index:

The term harvest index was proposed by Donald (1992) as the ratio of the grain yield to the aerial part of the biological yield. Harvest index by definition is a factor less than unity from 0 to 0.99, but can also be expressed in percent.

Screening of T3 sunflower transgenics for temperature stress tolerance using Temperature Induction Response (TIR) technique.

Temperature induction response (TIR) technique has been developed to identify thermo- tolerant lines. For the development of tolerant lines for any type of stress, exploitation of the genetic variability is inevitable. TIR technique is a potential technique to identify the genetic variability in high temperature tolerance. According to this technique, the seedlings are exposed to an optimum induction temperature, before exposure to lethal temperature and survival and growth after alleviation from lethal temperature will be determined. This phenomenon is called induced or acquired thermo tolerance. Genotypes with high survival and growth at the end of the recovery periods are selected as thermo-tolerant lines.

Selected 32 genotypes in the previous experiment were used for TIR techniques. Sunflower seeds were kept for germination in aluminum plates. Earlier Senthil kumar has standardized the TIR protocol in sunflower, thus I followed the same protocol for my study. As an absolute control one set of two days old seedlings were kept at 300C (room temperature). Another set of two days old seedlings were kept inside the incubator (Plate 9) with temperature and humidity control and subjected them to gradual increase in induction temperature from 280C to 450C for 5hrs. Immediately after induction temperature, seedlings were exposed to a challenging temperature of 490C for 2hrs and 30 min. Third set of seedlings were directly exposed to lethal temperature of 490C for 2 hrs and 30 min to study and compare the effect of gradual induction with that of direct exposure to lethal stress. After the treatment period, the seedlings were allowed to recover at room temperature for 72 hrs (Plate 10).

As most of the stresses lead to the creation of oxidative stress, it is imperative to look for the oxidative stress tolerance also. Here the oxidative stress was created in the seedling using menodione, a redox cyclic compound. As a control, 2 days old seedlings of almost uniform size were made to grow in petriplates impregnated with the distilled water. Another set with the 5ml of 5mM menodione for 3hrs (for induction) and later transferred to another plate impregnated with 20mM menadione (lethal concentration) for 24 hrs. Another set of seedlings were directly put to lethal concentration of menadione (20mM) for 24 hrs following which, the seedlings from both the treatments were washed thoroughly in distilled water and put for recovery for 3 days in petriplates impregnated with distilled water.

Observations

Percent seedling survival.

Initial root length in cm.

Total seedling length (cm)

Percent reduction in recovery growth was calculated by dividing the difference of the growth of the control (C) and the treatment (T) by the growth of the control and expressed in percentage.

C - T

Percent recovery growth =

C X 100

Molecular characterization of putative sunflower transgenics

Genomic DNA extraction by CTAB method

Leaf materials from salt tolerant and wild type plants were collected and total genomic DNA isolation was carried out using CTAB method (Doyle and Doyle 1990) with some modification. The plant material (100 mg) was ground in liquid nitrogen and homogenized in 750 µl of pre-warmed (65 °C) DNA extraction buffer [2% CTAB; 100 mM Tris-HCl, pH-8.0; 20 mM EDTA, pH-8.0; 1.4 M NaCl; and 0.2% -mercaptoethanol (added in situ before DNA extraction)] and further incubated at 65 °C in water bath for 45 min. An equal volume (750 µl) of chloroform : isoamylalcohol (24 : 1) was added after cooling it to room temperature and centrifuged at 20 °C for 30 min at 6000 rpm. To the supernatant (900 µl) two-third volume (600 µl) of chilled isopropanol was added and incubated at -20 °C for 1-2 hr. The mixture was later centrifuged at 8000 rpm for 10 min at 20 °C and the resulted pellet washed with 250 µl of 70% ethanol and centrifuged again (10 min, 8000 rpm, 20 °C). The supernatant was carefully discarded; the pellet air dried and then resuspended in 100 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH-8.0) and stored at room temperature. To this mixture 2 μl RNase A (2 mg/ml) was added and incubated at 37 °C for 1 hr. The supernatant was mixed with an equal volume (100 µl) of chloroform : isoamylalcohol (24 : 1) and centrifuged for 5 min at 10000 rpm. To the resulting supernatant, twice the volume of absolute ethanol was added and incubated at -20 °C for 1-2 hr. The mixture was centrifuged (10 min, 12000 rpm, 20 °C) and the pellet (genomic DNA) washed twice in 70% ethanol, air dried and resuspended in 50 μl TE buffer (1 mM) and stored at -20 °C.

Genomic DNA was checked on agarose gel (0.8 %) electrophoresis and then used for PCR amplification in an Eppendorf Master Cycler Gradient at the standardized annealing temperature.

PCR analysis of transformants

The presence of transgene in the transformants was analyzed by DNA amplification. Polymerase chain reactions were carried out using EcNAC1, hpt II and the combination of CaMV35S promoter forward-gene specific reverse primers. The Amplification was performed in the total volume of 15 μl containing 1.5 μl of 10X buffer (Fermentas, U.S.A), 1.5 μl of 2 mM dNTP, 1 μl of genomic DNA (100 ng), 1 μl of (5 pmol ) each forward and reverse primer, and 1μl (2.5 U/μl) unit of Taq polymerase. PCR were carried out with a Eppendorf thermal cycler by using initial denaturation at 94oC for 5 min followed by 30 cycles consisting of 94oC for 30 sec, 56oC (56oC for EcNAC1, 58oC for hpt II and CaMV 35S combination primer) for 30 sec and 72oC for 90 sec, a final extension step consisting of 72oC for 10 min. (The primer details are mentioned in Appendix I).

The PCR samples were analyzed on agarose gel electrophoresis. The standard DNA ladder was used to determine the presence of expected product size (Gene Ruler DNA ladders, MBI-Fermentas).

Restriction Digestion Analysis

The amplified EcNAC1 gene products were purified by PCR purification kit (Qiagen, Germany) according to the manufacturer's instruction and the gene product was digested with Sac I restriction enzyme (Fermentas, USA). The restriction digestion were performed in the total volume of 20 μl containing 2 μl of gene specific product, 2 μl of Sac I buffer B (Blue) 1x, 1 μl of Sac I restriction enzyme and 15 μl of nuclease free water. The digestion was carried out for 1 hour and the resulting digested products were resolved on 1% agarose gel.

Sub-cloning of EcNAC1 into expression vector

Bacterial strain and vector plasmid

Escherichia coli DH 5_ and BL21 (DE3) pLysS and the protein expression vector pGEX-4T1 (GE, USA) were made available from Department of Animal Science, Central University, Hyderabad, India. The pTZ57R/T vector harboring EcNAC1 transcription factor gene was available in the Department of Crop Physiology, University of Agricultural Sciences, Bangalore, India.

Primer designing

The gene sequence of EcNAC1 obtained from the National Center for Biological Information (URL: www.ncbi.nlm.nih.gov) and the gene specific primers were designed for the coding sequences of the gene using the software FASTPCR programme. The gene specific primers for EcNAC1 were designed with restriction sites, BamHI at 5' end and EcoRI at the 3' end. The primers were synthesized as desalted oligos from Sigma Aldrich.

PCR amplification

The full-length EcNAC1 gene was amplified with gene specific primers with BamH I and EcoRI restriction sites. Amplification was performed in a total volume of 50 μl containing 5 μl of 10X PCR buffer (Fermentas, USA), 5 μl of 2 mM of each dNTPs, 1 μl of T/A plasmid harbouring EcNAC1 gene (100 ng), 5 picomol of each forward and reverse primer, and 1μl (2.5 U/μl) unit of Pfu polymerase (Fermentas, USA). PCR was carried out in an Eppendorf thermal cycler using initial denaturation at 94°C for 5 min followed by 30 cycles consisting of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 90 sec, with a final extension step consisting of 72°C for 10 min. The PCR product was purified by PCR purification kit (Qiagen, Germany) according to the Manufacturer's instructions. Further pGEX 4T1 expression vector and amplified gene fragment was digested with BamH I and EcoRI.

Ligation

The linearized pGEX 4T1 expression vector was ligated with EcNAC1 gene fragment. The ligation reaction mixture was carried out at the final volume of 15 μl, which comprised of 1.5 μl T4 DNA ligase buffer (10X), '4' μl digested expression vector, 5 U of T4-DNA ligase (MBI-Fermentas) and '2' μl of DNA insert. The reaction was incubated at 18 °C for overnight.

Preparation of competent E. coli (DH5-10C) cells

The E. coli strain DH5-10C was used to prepare competent cells for routine transformation experiments to multiply different plasmids used in the current study. From a freshly streaked LB plate (Luria Bertani: yeast extract - 5 g/L; tryptone - 10 g/L; NaCl -10 g/L; pH - 7.0; Agar - 1.5%), a single colony was inoculated into 3 ml LB broth and grown overnight at 37 °C on a rotary shaker at 200 rpm. The overnight grown culture (1 ml) was inoculated to a sterile 1 L flask containing 200 ml of LB medium and was grown on a rotary shaker at 200 rpm. The growth of the culture was monitored every 30 min by measuring the OD600 (UV 2450, Shimadzu Corporation, Kyoto, Japan). When the OD reached to around 0.4, the culture was cooled on ice bath for 30 min. The cells were always kept on ice in subsequent steps. The cells were pelleted by centrifuging at 3000 rpm for 10 min at 4 °C. The pellet was resuspended in 10 ml of pre-chilled 1 mM CaCl2 and centrifuged at 3000 rpm for 7 min at 4 °C. The supernatant was poured off and the pellet was resuspended in fresh 10 ml 1 mM CaCl2. Cells were incubated on ice for 30 min and centrifuged at 3000 rpm for 5 min at 4 °C. Supernatant was discarded and the pellet was resuspended in 2 ml of CaCl2 and then 100 µl aliquots were dispensed into prechilled sterile tubes and tubes were transferred to -70 C freezer for storage.

Transformation of plasmid constructs into E. coli (DH5-10C) by KCM method

Around 25-50 ng of plasmid DNA or 1 µl of ligated product was mixed with 100 µl competent cells and incubated on ice for 15 min. Heat shock was given at 42 °C for 90 sec. Later 1 ml LB medium (was added to the transformed cells and further incubated at 37 °C (250 rpm) for 1 hr. Aliquots (100-200 μl) of the diluted cells were spread on appropriate selection plates and incubated at 37 °C overnight.

Screening of recombinant clones

The recombinant clones either in expression vector was initially selected on (50 µg/ml ampicillin) antibiotic media. The survived colonies were further tested for their recombinant nature by colony PCR using gene specific primer. A typical colony PCR was performed similar to that of a standard PCR except that the initial denaturation was carried out for 5-7 min.

Colony PCR

PCR was performed to directly analyze the positive transformants of EcNAC1 gene using the gene specific primers. Total 20 L reaction volume, PCR cocktail was prepared consisting of PCR Buffer, dNTPs, MgCl2, primers and Taq polymerase and colonies were resuspended individually into 20 L of the PCR cocktail and simultaneously the colonies were patched onto a separate plate to preserve it for future use. The reaction was incubated in a thermo cycler for 5-7 minutes at 94C to lyse the cells and inactivate nucleases. Amplification was done for 25-30 cycles (denaturation at 94C for 1 minute, annealing for 1 minute at 56C and extension at 72C for 1 minute). Final extension was done at 72C for 10 minutes. The amplified product was resolved on a 0.8 % agarose gel to confirm the presence of the gene.

Plasmid DNA isolation

The plasmid DNA was isolated by alkaline-lysis method (Sambrook et al., 1989) with some modifications. The bacterial colony (E. coli) containing the appropriate plasmid was inoculated in 3 ml LB medium with appropriate antibiotics and allowed to grow for 16hr at 37 °C and 200 rpm. The cells were centrifuged (5000 rpm, 5 min, 4 °C) and the pellet was resuspended in 200 μl of solution I (25 mM Tris, pH - 8.0; 10 mM EDTA and 50 mM glucose) and further incubated on ice for 10 min without shaking. 200 μl of solution II (0.2 M NaOH and freshly prepared 1% SDS) was added to the suspension, carefully mixed and further incubated for 10 min on ice. In order to obtain high quality plasmid DNA, 150 μl of solution III (5 M potassium acetate (60 ml), glacial acetic acid (11.5 ml) and water (28.5 ml)) was added to the suspension and carefully mixed to avoid breaking of DNA. The mixture was again incubated on ice for 10min and centrifuged at 12000 rpm for 15 min at 4 °C. The supernatant, which contains the plasmid DNA was carefully taken and mixed with 1μl of RNase A (10 mg/ml) and incubated at 37 °C for 45 min. Further, equal volume of tris saturated phenol : chloroform (1 : 1) was added, vortexed and centrifuged at 12000 rpm for 10 min at 4 °C. To the supernatant equal volume of chloroform : isoamylalcohol (24 : 1) was added and centrifuged at 12000 rpm for 10 min. The plasmid DNA in the supernatant was then precipitated with 0.1 volume of 3M sodium acetate (pH 5.2) and two volumes of ethanol and kept at -70 °C for 1 hr. The plasmid DNA was recovered in the pellet after centrifugation (12000 rpm for 10 min at 4 °C). The plasmid DNA was washed twice in 70% (v/v) ethanol and air dried at room temperature. The dried pellet was dissolved in 30 μl of sterile distilled water and stored at -20 °C until further use. A test gel (0.8% agarose gel) of 10 μl of plasmid prep was made to monitor the purity of extraction. The OD of sample was measured at 260 nm to determine the DNA concentration.

Restriction digestion analysis

The plasmid DNA was subjected to single and double restriction enzyme analysis. The total size of the construct (vector + EcNAC1 gene) was detected by single restriction digestion with EcoRI and double digestion was carried out with BamHI and EcoRI for insert release. The resulted product was separated by agarose gel electrophoresis. The restriction digestion reactions were carried out at 37ï‚°C for 2 hours. The enzymes and the buffers used for restriction reactions were obtained from MBI-Fermentas.

Separation of DNA on agarose gel

DNA fragments were separated using horizontal gel electrophoresis. Depending on the requirement 0.8 to 1% agarose solutions were prepared in 1X TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH-8.0) by heating in a microwave oven. 0.5 μg/ml ethidium bromide was used for staining. DNA samples were mixed with 0.1 volume loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol and 15% glycerol) and separated electrophoretically. The detection of the DNA fragments was carried out under UV-transilluminator (302 nm) and photographed using a gel documentation system (Biorad, USA).

Sequencing and Multiple alignment of EcNAC1 gene

Sequencing of pGEX 4T1-EcNAC1 was done using the T7 promoter specific forward primer and sequence analysis was done using multiple alignment software.

Transformation of pGEX 4T1-EcNAC1 plasmid constructs into expression host (E. coli BL21 (DE3) pLysS) by calcium chloride method

The E. coli strain DH5-10C was used to prepare competent cells for routine transformation experiments to multiply different plasmids used in the current study. From a freshly streaked LB plate (Luria Bertani: yeast extract - 5 g/L; tryptone - 10 g/L; NaCl -10 g/L; pH - 7.0; Agar - 1.5%), a single colony was inoculated into 3 ml LB broth and grown overnight at 37 °C on a rotary shaker at 200 rpm. The overnight grown culture (1 ml) was inoculated to a sterile 1 L flask containing 200 ml of LB medium and was grown on a rotary shaker at 200 rpm. The growth of the culture was monitored every 30 min by measuring the OD600 (UV 2450, Shimadzu Corporation, Kyoto, Japan). When the OD reached to around 0.4, the culture was cooled on ice bath for 30 min. The cells were always kept on ice in subsequent steps. The cells were pelleted by centrifuging at 3000 rpm for 10 min at 4 °C. The pellet was resuspended in 10 ml of pre-chilled 1 mM CaCl2 and centrifuged at 3000 rpm for 7 min at 4 °C. The supernatant was poured off and the pellet was resuspended in fresh 10 ml 1 mM CaCl2. Cells were incubated on ice for 30 min and centrifuged at 3000 rpm for 5 min at 4 °C. Supernatant was discarded and the pellet was resuspended in 2 ml of CaCl2 and then 100 µl aliquots were dispensed into prechilled sterile tubes and tubes were transferred to -70 C freezer for storage.

Induction of pGEX 4T1-EcNAC1 plasmid transformed E. coli BL21 (DE3) pLysS cell with IPTG

Expression of recombinant protein in E. coli BL21 (DE3) pLysS cells was studied in the positive

clones carrying recombinant plasmid pGEX 4T1-EcNAC1. The positive clones were grown in LB medium containing ampicillin (50 μg/ml) and chloramphenicol (34 μg/ml) at 37°C, and when A600nm reached to 0.4-0.5 the culture was induced with 0.4 mM isopropyl-_-D-thiogalactopyranoside (IPTG) for 4 h at 30°C. To identify and localize the EcNAC1 protein produced by E. coli, the cytosolic and pellet (insoluble fraction containing inclusion bodies) fractions of the bacterial culture were electrophoresed through 12.5% SDS polyacrylamide gel along side a standard protein molecular weight markers (Fermentas, USA). The SDS-PAGE gel was stained with Coomassie brilliant blue in Methanol:Acetic acid:Water (45:10:45) and destained in the same solution. The stained bands were visualized under an illuminator.

SDS-PAGE

Clean and dry the glass plates and spacers, then assemble them properly. Mix the components for the resolving gel with ammonium persulphate and TEMED. Pour the resolving gel mixture into the gel plates to a level 2 cm below the top of the shorter plate. Place a layer of deionised distilled water or isobutanol over the top of the resolving gel to prevent meniscus formation in the resolving gel. Allow resolving gel to stand 30 min at room temperature. Drain the deionised distilled water from top of the resolving gel. Rinse with deionised distilled water, drain, and wick any remaining deionised distilled water away with a filter paper. Mix components for stacking gel. Pour stacking gel solution into gel plates on top of the resolving gel, so that gel plates are filled. Insert comb to the top of the spacers. Allow gel to stand for at least 1 hr at room temperature. Remove the comb without distorting the shapes of the well and wash the wells with deionised distilled water using a syringe. Add freshly prepared 1x running buffer to both chambers of the apparatus. Load the prepared samples (soluble and insoluble fraction from bacterial cell lysate) into the wells of the gel. Run the gel at 90 V until the dye front migrates into the resolving gel (~30 min), and increase to 150 V until the dye front reaches the bottom of the gel.

Remove the run gel from the aparatus and remove the spacers and glass plates. Place the gel into a small tray. Add 20 ml staining solution and stain for more than 30 min with gentle shaking. Pour off and add 5 ml destain solution and destain for 1 min with gentle shaking. Pour off and discard the destain solution. Add 30 ml of destain solution. Destain with gentle shaking until the gel is visibly destained (> 2 hr). Pour off and discard the destain solution. Rinse with deionised distilled water for 5 min with gentle shaking. Dry the gel on the gel dryer at 60°C for 1 hr with a sheet of Whatman filter paper below the gel and a piece of Seran wrap over the gel.

Electrophoretic elution of fusion proteins from gel

In order to extract the protein from the polyacrylamide gel, the method of electro-elution was

applied using dialysis membrane [4,5]. Protein band with 66 kDa size was excised and cut into

small fragments. The stain CBB-R250 from the gel fragments was removed as per the method

described [6]. Briefly, destaining solution containing 50% isopropanol and 1.5% SDS in gel running 461 buffer was added to gel pieces in glass tube and the tubes were capped with parafilm. Tubes were placed in 37°C water bath set for overnight without agitation. After cooling at room temperature, the liquid was removed and the gel fragments containing the appropriated protein were used for electrophoretic elution. Preparative SDS-PAGE electrophoretic elution was done as per the method described earlier [7]. Briefly the gel fragments were equilibrated twice in 0.1 M Tris-HCl buffer (pH 6.8) and 1.0% solution of 2-mercaptoethanol for 20 min. A final equilibration of the gel fragments in 0.1 M Tris-HCl buffer (pH 6.8) containing 1.0% (w/v) SDS was performed. The gel fragments were then placed in a dialysis tube with tris-glycine buffer containing SDS (25mM Tris, 192 mM glycine, and 0.1% SDS) and electroelution was performed at 50 V for 10 h at 4°C in Tris glycine buffer containing 0.1% SDS (pH 8.3). At the end of electrophoretic elution, the polarity of the electrodes was changed for 1 min in order to release the absorbed protein on the dialysis tubes. The electroeluted protein sample was again dialyzed twice in PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4).

Western blot analysis

A western blot defined as the electrophoresis of the antigen followed by its subsequent transfer to nitrocellulose paper and incubation with specific antibody and then with labelled secondary antibody.

Procedure:

Once SDS-PAGE was completed, Whatmann no 1 filter paper was cut to the same dimension as that of the gel. Usually 12 such pieces were cut out and presoaked in the transfer buffer (0.025 M Tris- Glycine buffer, pH 8.8 and 0.1% SDS). The nitro cellulose membrane was also cut exactly to the same size as that of the gel and soaked in transfer buffer prior to use. The gel was soaked in the transfer buffer for equilibration. Preparation of the transfer stack: 6 pieces of filter paper were stacked over one another followed by the nitrocellulose membrane, gel and over the gel another 6 pieces of filter paper were placed one over the other. Before placing the gel, suitable marks are made to indicate the position of the marker lane. Eletroblotting should be done for 2 hrs at 50 volts and 45mA current. The membrane is kept at 4°C overnight. The membrane is then placed in the blocking solution for 1Hr .(The blocking is done so that the area of the nitrocellulose where no protein has been transferred is occupied by milk powder, which in turn prevents non-specific binding of the antibody). After 1 hr blocking solution was poured out and washed the nitrocellulose membrane with PBST wash solution. 3 washes of 10 min each should be done.

The nitrocellulose membrane was placed in the primary antibody solution (supplied by R.K. Jain, IARI, New Delhi) and kept at room temperature on a rocker for 1 hr. The membrane was washed with PSBST wash solution. 3 washes of 10 min each was carried out, changing the wash solution after each wash. After washing, the membrane was placed in the secondary antibody solution (supplied by Genei, Bangalore) for 1 hr at room temperature on a rocker. Later the membrane was washed with PSBT wash solution. 3 Washes of 10 min each was carried out, changing the wash solution after each wash. After washing, the membrane was placed in substrate solution which provides colour only where there is binding of the primary and secondary anti body. Usually colour develops within 30 min. The reaction was carried out in dark conditions. The reaction was stopped by rinsing the membrane with water and dried under dark conditions. The membrane should not be exposed to light as the bands fade out. Photograph was taken for permanent record.

Polyclonal antibody production

Antibodies against electroeluted fusion protein (GST-EcNAC1) were raised in a New Zealand white rabbit. The purified protein (100 μg) in 1.0 ml of PBS was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously at 6 to 8 sites at the back of the neck, of an adult healthy rabbits of body weight 2.5-3.0 Kg. Three-booster injections with 50 μg each with Freund's incomplete adjuvant were given at every three weeks intervals. The animals were bled two weeks after each injection and the serum was separated, aliquoted and stored at -20°C.

Western blot analysis to confirm EcNAC1 protein in Finger millet

SDS-PAGE separated proteins were electroblotted onto a nitrocellulose membrane at a constant

current of 90 mA at room temperature for 1 h. After blocking the leftover free sites with blocking

buffer (PBS containing 5% (w/v) skimmed milk powder, the membrane was incubated with the

antiserum antibodies raised against GST-EcNAC1 (1:1000 diluted in PBS containing 0.05% Tween-20 and defatted milk powder) at 37°C for 2 h. After washing three times, goat-anti-rabbit IgG conjugated with HRP (diluted 1:5000 in blocking buffer) was added and incubated for 1 h at room temperature and the blot was developed with DAB (3,3'Diaminobenzidine) substrate.

Expression analysis - DAC-ELISA (Directed antigen coated ELISA)

The samples of different transformed and non transformed sunflower plants from the green house were collected and tested by direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) protocol.

Protein sample preparation

Total 100 mg finger millet leaf sample grind to homogenate using pestle and morter with 500 µl of Phosphate buffer pH 7.0. The homogenate solution centrifuged at 13,000 rpm for 30 min at 4oC. After centrifugation the supernatant was transferred to new eppendorff tube and placed on ice for further use.

DAC-ELISA

Total 100 μl of antigen diluted with carbonate buffer (1:1 dilution) coated to wells of a ELISA microtiter plate and incubated for 2 hours at room temperature. Remove the coating solution and wash the plate three times by filling the wells with 200 μl PBS. The remaining drops are removed by patting the plate on a paper towel. Block the remaining protein-binding sites in the coated wells by adding 200 μl blocking buffer (5% skimmed milk powder) and incubate for 2 h at room temperature, after incubation wash the plate three times with PBST. Then add 100 μl of 1:1000 diluted primary antisera (anti-EcNAC1) to each well and incubate for 2 hours at room temperature.Wash the plate three times with PBS. Add 100 μl of 1:5000 diluted HRP conjugated secondary antibody, incubate for 1-2 hours at room temperature. Wash the plate three times with PBS. Finally add 100 μl TMB solution to each well, incubate for 30 min, add equal volume of stopping solution (2 M H2SO4) and read the optical density in ELISA reader (Compaq Imaging System) at wavelength 450 nm.

Estimation of total soluble protein: The protein concentration in an extract determined by the dye-binding assay of Bradford. 5 µl of the antigen extract and 295 µl of the Bradford reagent added to 96 well plate and blue colour measured at the wavelength 595nm.

The standard curve prepared using BSA (2.0-10.0 ng/µl) solution. (5 µl of the BSA and 295 µl of the Bradford reagent added to 96 well plate and blue colour measured at the wavelength 595nm).

Expression analysis - Reverse-Transcription Polymerase Chain Reaction (RT-PCR) analysis

Isolation of total RNA

Leaf material (1g) was frozen in liquid nitrogen and ground to fine powder, 10ml of extraction buffer was added (0.1M Tris HCl, pH 9.0, 0.25M sucrose, 0.2M NaCl and 10mM MgCl2) and homogenized. To the extract 10ml of water saturated Phenol: Chloroform (1:1 v/v), 1ml of 1mM Sodium EDTA and 1ml of 10 per cent SDS were added and homogenized. The extract was then transferred to centrifuge tubes and 144l of Mercaptoethanol was added and incubated at 40 C for 20min, under constant shaking. The extract was then centrifuged at 16,000 rpm for 30 minutes and supernatant was separated into fresh centrifuge tube. Equal volume of Phenol: Chloroform mixture was added to aqueous phase, vortexed and spun at 15,000 rpm for 15min at 4oC. Aqueous phase was transferred to fresh centrifuge tube and equal volume of Chloroform: Iso-amylalcohol (48:2) was added and centrifuged at 16,000 rpm for 15 min at 4oC. Aqueous phase was transferred to fresh centrifuge tube and 8M LiCl2 was added to a final concentration of 3M and incubated at 4oC for 20 hrs. After incubation the extract was centrifuged at 16,000 rpm for 30 min at 4oC and the supernatant was discarded. The precipitate was washed with 5ml of 2M LiCl2 and with 5ml of 75 per cent ethanol by centrifuging at 15,000 rpm for 20min at 40C. The pellet was air dried and dissolved in 100l of DEPC treated water. The extracted RNA was quantified by using nanodrop, the purity was analyzed by OD 260/OD280 ratios as well as visualizing on an Agarose (1%) denaturing gel.

Preparation of RNA gel

The total RNA was separated on Agarose gel (1%) containing formaldehyde. About 20g of RNA was mixed with RNA gel loading buffer and incubated at 650C for 10 minutes before loading. The separated RNA was visualized with the help of gel documentation unit (Bio Rad).

Synthesis of cDNA from the extracted total RNA.

Initially, a 10 l reaction mixture [consisting of 5 g of total RNA, 1 l of Oligo (dT)18 primer (10 pmol/l) and RNase free sterile distilled water] was prepared in a 0.2 ml PCR tube and incubated at 70 °C for 5 min (to remove any secondary structure) and immediately placed on ice. Subsequently, 10 l of another reaction mixture [consisting of 2 l of dNTPs (10 mM), 4 l of M-MuLV RT buffer (5X), 50 U of M-MuLV Reverse Transcriptase (MBI-Fermentas) and sterile water] was added and briefly spinned. The resulting 20 l reaction mixture was incubated at 42 °C for 1 h. Further, the enzyme was inactivated by incubating the reaction mixture at 90 °C for 5 min.

Polymerase chain reaction

The PCR was carried out in a 20 l reaction mixture containing 1 l of cDNA, 2 l of Taq assay buffer (10X), 1 U of Taq DNA polymerase (Bangalore Genei), 1 l of MgCl2 (25 mM), 1 l of dNTPs (2 mM), 5 pmol of forward (Individual gene specific primer: Appendix) and reverse primer (Individual gene specific primer: Appendix) and sterile water. Amplification was performed under standardized conditions in Master cycler Gradient (eppendorfAG, Germany). The PCR amplified products were resolved on agarose (1.0 %) gel using 1X TAE buffer stained with ethidium bromide and were visualized using UV-transilluminator and documented.

Response of transgenic tobacco seedlings under moisture stress

The in vitro grown tobacco plants transformed with either pG-4XABRE-EcNAC1 or pG-CaMV 35S-EcNAC1 were hardened and transferred to pots containing soil mixture. The plants were grown for 20 days under ambient conditions. Moisture stress was imposed by withholding water for 15 days and the stress response was seen a week after rehydration.

Statistical Analysis:

The data obtained in the present study were computed and analyzed statistically following the Duncan Multiple Range Test using MSTATC Software.

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