Plant tissue collection
Immature jatropha (Jatropha curcas) seeds, at 4-5 weeks after fertilization were selected for total RNA extraction. Rice (Oryza sativa) seeds 21 days after fertilization and mustard (Brassica spp.) seeds 20 days after fertilization were also used. Before grinding, the kernels of immature jatropha seeds were separated from the seed shell, whereas for rice seed husks were removed using sterilized scissors or forceps and stored at -80°C until use. Mustard seeds were removed from pods and used as such.
Total RNA extraction method
About 0.5 g of each seed sample was ground in liquid nitrogen using oven baked RNase-free mortar and pestle and the seed powder was then transferred to a pre-chilled 50-mL polypropylene (Falcon) tube. Five mL of pre-heated (65°C) total RNA extraction buffer {2% (w/v) CTAB (Sigma), 2% (w/v) polyvinylpyrrolidone (PVP-40) (Sigma), 100 mM Tris HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, 0.1% spermidine (Sigma) and 2% β-mercaptoethanol} was added to the powdered seeds in each tube and samples were incubated for 30 min at 65°C in a water bath. The samples were placed on a vortex every 5 minutes to help tissue disruption and RNA extraction in the buffer. After incubation, an equal volume of Chloroform: Isoamylalcohol (24:1) was added to each sample in a fume hood and samples were mixed with a vortex for 30 seconds. Thereafter the samples were centrifuged at 10,000 g for 20 minutes at 4°C. The aqueous supernatant (1 ml/tube) above the white phase was carefully transferred into 2.0 mL RNase-free microcentrifuge tubes and an equal volume of Chloroform: Isoamylalcohol was added, mixed with a vortex and centrifuged in a desktop centrifuge at 10,000 g for 10 minutes at 4°C. Without touching the white layer, the supernatant (1.0 ml) was distributed to Rnase free1.5 mL microcentrifuge tubes and 0.5 mL of 96-100% ethanol was added. The supernatant-ethanol mixture was immediately loaded onto RNA binding columns (0.75 mL/column) skipping filtration step (Qiagen RNA Mini extraction kit or any other similar kit) and spun at 10,000 g for 30 seconds at room temperature. Leftover samples were loaded on the same columns to process the entire sample. The kit protocol was followed in subsequent steps to wash and desalt the samples bound with the silica membrane of the column. Finally, the RNA from each column was eluted using 50 μL of RNase free water and stored at -80°C.
The quality of RNA was checked using a spectrophotometer (NanoDrop, Technologies Inc.) at two wavelength ratios of A260/230 and A260/280 nm. The integrity of total RNA was determined by running samples on 1.2% denaturing agarose gel (Qiagen, RNeasy Mini Handbook). The intensity of 28S and 18S bands was quantified with Molecular Imaging software version 5.1 (Kodak, Rochester, NY). Aliquots of RNA were stored at -80°C.
cDNA synthesis and cDNA library construction
Poly(A)+ RNA was purified from total RNA of immature jatropha seeds using the Oligotex® Midi mRNA kit (Qiagen, Germany), dissolved in RNase-free water, quantified with spectrophotometer (NanoDrop, Technologies Inc.), and stored at -80°C. The cDNA library was constructed using CloneMiner™ cDNA Library Construction Kit (Invitrogen). The first strand of cDNA was synthesized using 5 μg poly(A)+ RNA and converted into double strand cDNA (ds cDNA) containing att B sequences on each end followed by ligating att B1 adapter to the 5' end of cDNA. The cDNA was size fractionated by column chromatography to remove excess of primers, adapters, and small cDNAs. A non-radio labeled method was used to determine the cDNA yield. About 75-100 ng of cDNA obtained from different pooled fractions was used in site-specific recombination and att B-flanked cDNA was cloned into an att P-containing donor vector (pDONR™ 222). The BP reactions were transformed into ElectroMAX DH10B T1 phage resistant cells using an electroporator (Life technologies) and the transformed cells were plated on kanamycin (50 μg/mL) added LB agar media. Twenty positive clones were picked for verification of cDNA inserts. The mini-prepared plasmids were digested with BsrG 1 enzyme (New England Biolabs) and electrophoresed on 1% agarose gel to determine average insert size of cDNA.
Amplification of KAR gene using jatropha cDNA
Primers from the Arabidopsis 3-ketoacyl-acyl carrier protein reductase, (AT1G24360) (KAR) involved in Fatty acid biosynthesis were selected from the GenBank database [20] for RT-PCR on jatropha seed cDNA. The amplification program for PCR consisted of an initial denaturation step at 94°C for 2 min, followed by 35 cycles of 30 s denaturing (94°C), 45 s annealing (60°C), 1 min elongation (72°C), and a final extension at 72°C for 5 min. The amplified PCR product was visualized on agarose gel [21], and extracted from the gel using the QIAquick PCR purification kit (QIAGEN) to probe RNA blot. The PCR product was cloned into the Teasy vector (Promega) and sequenced using BigDye termination method with AB1377 sequencer (Applied Biosystems, Foster City, CA, USA). The sequenced product was confirmed by aligning with the Arabidopsis KAR nucleotide sequence at NCBI using BLAST.
Northern blot analysis
For Northern blotting, 15 μg of RNA was isolated from immature jatropha seeds and leaves and fractionated on 1.2% agarose-formaldehyde denaturing gel (Qiagen RNeasy Mini handbook). The RNA was blotted onto Hybond-N+ nylon membranes (Amersham Pharmacia) and stained for visualization of the RNA bands [17]. The KAR cDNA probe generated using RT-PCR was labelled with [32P]-dCTP (GE Healthsciences). Pre-hybridization was for 3 hours and hybridization was for 16 hours at 65°C (Techne, Staffordshire UK). Filters were washed first (20 min) in buffer A (2 × SSC + 0.1% SDS) and then Buffer B (20 min) in (1 × SSC + 0.1% SDS) and lastly (30 min) in buffer C (0.5 × SSC + 0.1% SDS) at 65°C. The bound probe was detected by exposing filters to KODAK Biomax MS Autoradiography Film using exposure cassettes at -80°C.