Bacterial strains and plasmids
The Bt and bacterial strains were taken from “The Microbial Type Culture Collection and Gene Bank” (MTCC),Institute of Microbial Technology, Chandigarh, India. Vectors, plasmids used in this study were picked up from Fermentas INC.
Isolation of cry1Ac gene from Bacillus thuringiensis:
The Bacillus thuringiensis culture was inoculated in LB broth medium and incubated overnight at 37°C on a shaker. Genomic DNA has been separated from the culture and used as PCR template according to reported method (Carozzi et al. 1991). The genomic DNA isolated from Bacillus thuringiensis used as template for PCR to amplify the full length cry1Ac gene using 5′–GGCCATATGGATAACAATCCGAACAT–3′ and 5′–CCCGGGCTATTCCTCCATAAGGAGTA–3′ as forward and reverse primers.The underlined regions in the primers indicate NcoI and SmaI restriction sites.The PCR product is electrophoresed in 0.8% agarose gel in 1X TAE buffer. The eluted PCR product of ~3537 bp is digested with NcoI and SmaI restriction enzymes and cloned between CaMV 35S promoter and poly(A) terminator of pRT100. The recombinant pRT100 vector harbouring cry1Ac was named as pRT100-cry1Ac. The recombinant clones of pRT100-cry1Ac were further confirmed by NcoI and SmaI restriction digestion. The cry1AC cassette (CaMV35S-cry1Ac-poly(A)) in pRT100-cry1Ac was excised using HindIII restriction enzyme and used for further ligating to APX cassette in the further steps.
Isolation of L-ascorbate peroxidase gene from rice plant
On the other hand, the fresh leaves of rice plant were selected and their total RNAs have been separated by making use of TRIzol reagent. The synthesis of the first strand of the cDNAs was done by utilizing Prime Script™ RT reagent kit with g DNA eraser (Takara, Japan) as per the instructions by the manufacturer. cDNA has been utilized as a template and PCR was done using 5′–GGCCATATGCTGATCTTTATCAGCTT–3′ and 5′–CCCGGGCTACTTGGTTTTCTTAGAAG –3′ as forward and reverse primers. The amplified ~516 bp fragment of L-ascorbate peroxidise gene was eluted from the agarose gel by freeze-thaw gel elution method using low-melting agarose (Sambrook and Russell 2001). The eluted ~516 bp of L-ascorbate peroxidase gene fragment was cloned between CaMV 35S and poly(A) of pRT100 vector at NcoI and SmaI restriction sites. The recombinant pRT100 clones were designated as pRT100-APX. The CaMV35S-APX-poly(A) cassette in pRT100-APX was excised using PstI restriction enzyme. Both the HindIII restriction fragments of cry1Ac cassette (CaMV35S-cry1Ac-poly(A) and APX cassette CaMV35S-APX-poly(A)) were ligated using DNA ligase and then sub-cloned at HindIII/ PstI site of pCAMBIA3300 vector containing bar gene as selectable marker. The resultant recombinant pCAMBIA3300-cry1Ac-APX (pCAMBIA3300-CaMV35S-cry1Ac-poly(A)-CaMV35S-APX-poly(A)) vector was mobilised into E.coli HB101 cells and were confirmed by HindIII and PstI restriction digestions. Later, recombinant pCAMBIA3300-cry1Ac-APX was mobilised into Agrobacterium (LBA4404) by tri-parental mating by utilizing helper plasmid PRK2013 (Ramesh et al. 2004).
Genetic transformation and generation of transgenic plants
Rice cultivar BPT 5204 seeds obtained from Indian Institute of Rice Research (IIRR), Hyderabad (India), were used for genetic transformation experiments. LBA4404 harbouring recombinant pCAMBIA3300 (pCAMBIA3300-cry1Ac-APX) vector was employed for Agrobacterium mediated genetic transformation. Scutellum-derived calli of BPT 5204 seeds were co-cultivated with AgrobacteriumLBA4404 culture with an OD of 0.8. For selection of (pCAMBIA-cry1Ac-APX) transformants, co-cultivated calli is allowed for two rounds of selection on medium with 8 mg/L phosphinothricin. Actively growing calli is selected and moved to the proliferation medium. After 10 days, the proliferated calli is transferred onto shooting medium then to rooting medium. About 40-50 days old putative transformants were tested for their tolerance to 0.25% basta.
PCR analysis of pCAMBIA-cry1Ac-APX transformants
Genomic DNA was isolated from pCAMBIA-cry1Ac-APX putative transformants as well as from untransformed control (UC) plants as per McCouch (1988). PCR analyses was doneutilizingcry1Ac gene specific forward 5′-AGTCCAATCGGAAAGTGTGG -3′ and reverse 5′-AAAATAGCCGCATTGACACC-3′ primers and APX gene specific forward 5′-ATCAGCTTGCTGGAGTGGTT-3′ and reverse 5′-GCCGTTGAACCATCTGATTT-3′ primers the PCR products have been further analysed on a 1.0% agarose gel. DNA from the untransformed plants acted as negative control and plasmid DNA as positive control.
Southern blot analysis of transgenic plants
Southern blot analysis was performed using 18 μg of genomic DNA, isolated from pCAMBIA-cry1Ac-APXtransgenic plants,digested with HindIII restriction enzyme. The DNA which has been digested was extracted on 0.8% agarose gel. Late after the denaturation and also neutralization, the DNA has been shifted to a H-N+ membrane (Amersham Pharmacia) and later cross-linking was performed through exposing to UV (1200 µJ for 60 s). The cry1Acregion and APX coding regions were radiolabeled with α-32P dCTP using ready to Go DNA labeling beads and used as a probe. After labelling,hybridization steps and washing of the membranes were carried out as per the instructions given by the manufacturer.
Generation of homozygous transgenic lines
To generate homozygous lines of pCAMBIA-cry1Ac-APX transgenic lines seeds obtained via putative transformants were put to growth on MS basal medium along with 6 mg/L phosphinothricin (PPT). After 10 days, the resistant (T2) seedlings were moved to pots and grown. The seeds obtained from T2 plants were later germinated on the MS medium containing 6 mg/L PPT for selection of homozygous lines.
Functional validation of transgenic plants for Abiotic Stresses
Drought stress at seed germination and seedling stage
Seeds of homozygous lines were surface sterilized with 70 % (v/v) ethanol for 1 min followed by 0.1% (w/v) Mercuric chloride (HgCl2) for 4 min and then washed with sterile distilled water for three times. To assess the drought stress response, the sterilized seeds were dried on sterile filter papers and transferred onto MS basal medium supplemented with 250 mM mannitol. The seed germination rates were taken late after 10 days. For stress treatment at seedlings stage, seeds of homozygous lines were germinated in Hoagland solution at 30°C for 2 week. Later they have been transferred to fresh Hoagland solution containing 250mM mannitol for 7 days. After stress treatment, the seedlings have been shifted to fresh Hoagland solution for recovery. After 7 days, the survival rate, shoot and root length, and seedlings biomass have been registered.
Drought stress at the vegetative stage
Seeds of homozygous lines were grown on MS medium and then established in pots. 40-50 days old plants in pots were selected for drought stress treatment. Drought (with-hold of water) stress treatment was applied at the vegetative stage for 10 days at30°C. After 10 days of treatment, plants were supplied with water at regular basis for recovery. Data on antioxidants, chlorophyll, water content, reducing sugars, proline content was deduced.
Drought stress at the reproductive stage
Plants are allowed to grow under normal conditions till the inflorescence. At reproductive stage, drought stress treatment was imposed by withholding water for 10 days at 30°C. After 10 days of withholding water, the plants were supplied with water for recovery. The data on number of filled grains per panicle, total chlorophyll content, anti-oxidants activity were recorded. Five plants were used in each treatment and all the experiments were repeated thrice.
Total chlorophyll content
Total chlorophyll content is calculated as per Lichtenthaler and Wellburn. Twenty five mg of fresh leaf of treated and untreated samples of transgenic plants along with UCare incubated in 10 ml of 80% acetone and left in dark for about 48 hours. Later the absorbance rate has been calculated at 663 and 647 nm for chl a and chl b content, respectively.
This is carried out as per Bates (1973) documentation. One gram of fresh leaf from both transgenic, UC plants were broken down with 20 ml of 3% sulfosalicyclic acid (W/V) and the obtained homogenate has been centrifuged for around 10 minutes at 10,000 rpm. The supernatant is utilized for proline estimation. Reaction mixture with 2.0 ml of glacial acetic acid, ninhydrin reagent and supernatant was boiled to 110°C for about 1 hour. Late after the reaction mixture is cooled down, 4 ml of toulene was included and vortexed for 30 about seconds over a cyclomixer. The chromophore from aqueous phase has been collected, absorbance is measured 520 nm.The concentration of Proline was analysed from the standard curve and given in microgram of proline per gram of the fresh material.
Estimation of reducing sugars
Leaf tissues (100 mg) of transgenic and UC plants were freezed using liquid nitrogen and made into a powder. Reducing sugars of about 2 times were separated from the powder by using 80% ethanol at about 95°C. The supernatant is concentrated by drying at about 80°C for around 2 hours. The residue was later dissolved in distilled water and the reducing sugarsare used for estimating as per the available method (Miller 1959).
Extraction and estimation of antioxidant enzymes
The extraction procedure for both SOD and CAT is similar. Transgenic and UC plants leaf samples of 100mg were frozen by utilizing liquid N2 in order to avert proteolytic activity. The frozen tissues are broken down by utilizing 5ml extraction buffer with 100mM phosphate buffer (pH 7.5) and 0.5 mM EDTA and later centrifuged for about 15min at 12,000 rpm and 4°C.The supernatant was utilized used for the analysis of enzyme.
Estimation of Superoxide dismutase activity (SOD)
The activity of SOD was assessed by checking over the reticence of photochemical reduction of nitroblue tetrazolium. Later enzyme activity have been analysed by including100μl of enzyme extract to 3ml of the reaction mixture having 13mM methionine, 2μM riboflavin, 50mM potassium phosphate buffer (pH 7.8), 75μM NBT, and 0.1mM EDTA. After that thereaction mixture was illuminated with 5000lux light intensity. Later absorbance was checked at 560 nm by utilizing spectrophotometer. SOD activity is the amount of enzyme required to impede half rate of inhibition of the NBT reduction.
Catalase (CAT) activity
Leaf samples from stressed plants were collected and homogenized in 50mM phosphate buffer (pH7.0). The obtained homogenate was later centrifuged at 8000g, 20min, 4°C. The extract of Enzyme is added to the H2O2-phosphate buffer (pH 7.0), and time taken for the absorbance at about 240nm from about 0.45-0.40 was considered. The Catalase activity was analysed as per Aebi (1984) and protein estimation was carried out using Bradford reagent.
Malondialdehyde (MDA) content
The leaf tissues were allowed for homogenization in 5ml 0.1% TCA (trichloroacetic acid) and then sent for centrifugation at 5000 g, 10min. 500µl of the obtained supernatant is mixed with 4ml of 20% TCA having thiobarbituric acid (TBA) (0.5%). Later, mixture is heated up at around 95°C, 30min, and then cooled down on ice, later centrifuged at 5000 g, 15min, and then the absorbance was noted at 532 and 600nm. After excluding the non-speciﬁc absorbance, MDA activity was measured by utilizing the extinction coeﬃcient of 155mM−1 cm−1.
Relative water content measurement
To measure this, the leaves of transgenic and UC plants fresh weight was measured in relation to fresh leaf weight. Plants leaves were left at room temperature till no loss in weight (desiccated weight). Late after, the leaves were allowed to dry for about 24 hours, 70°C and the dry weights have been noted. The below is the formula used to measure RWC.
RWC (%) = (desiccation weight - dry weight)/ (fresh weight - dry weight) x 100.
Quantitative Real Time PCR (qRT-PCR)
For qRT-PCR analysis, RNA samples extracted from untransformed control(UC) and APX transgenic seedlings subjected to 200mM mannitol treatment for 24h along with unstressed plants were utilized for synthesizing first strand cDNA.qRT-PCR analyses were carried out using SYBR green master mix with Applied Biosystems 7500 real time PCR system programmed with 94ºC (1min), 59ºC (45 sec) and 72ºC (1min) for 30 cycles. The RT-PCR products obtained were analysed through the melt curve analysis to check the speciﬁcity of PCR ampliﬁcation. All the reactions were performed thrice and the relative expression ratio was calculated using 2-ΔΔct method employing actin gene as a reference. The primers used for qRT-PCR were tabulated in supplementary material (Table S1).
All of the obtained data were determined by utilizing Student’s t-test for statistical significance. **P <0.01 and *P <0.05 shows differences at 1%, 5% level of significance, when compared with wild-type of control.