Published on August 20, 2014
Communications in Plant Sciences (2237-4027) volume 2, issues 1-2, p. 5-9, Jan-Jun, 2013 6 Raghava et al. 2013. Herbivory induces differential gene expression … differentially in few tomato cultivars. Four genes chosen for study are involved in oxidative stress, protein processing, gene expression modulation and Jasmonate signaling pathways upon herbivory in plants. CORONATINE INSENSITIVE-1 (COI-1), an F- box protein, involved in regulating Jasmonate mediated signaling pathways during herbivore damage (Howe et al. 2004) and promotion of glandular trichome-based defenses (Lei et al. 2004). DNA binding protein (DBP) is a transcription factor that binds to AT-hook motif is being evaluated for modulation in the expression levels (Aravind and David 1998). Leucyl aminopeptidase (LAP), an exopeptidase, catalyzes the release of N-terminal residues from proteins and peptide is chosen for this study. LAP and LAP-like proteins in tomato are induced locally and systemically on herbivory (Mahagamasekera and Team 2001, Yong-Qiang et al. 1999, VanDoorn et al. 2011). A Peptidyl propyl isomerase (PPI), involved in oxidative stress and protein processing during proteolytic events of a cell, they are considered as protein folding catalyst (Björn et al. 2009) is also used for evaluation of gene expression levels. The expression levels of four transcripts were evaluated using semi-quantitative PCR which is a reliable tool being used in molecular studies recently (Jerry et al. 2006). The transcript levels of COI-1, LAP, PPI and DBP were analyzed in intact and herbivore, S. litura damaged leaves of S. lycopersicum cuv. All Rounder, Lakshmi and Shaktiman. MATERIAL AND METHODS Plant material and growing conditions . Twenty five day old plantlets of cultivar All Rounder, Lakshmi and Shaktiman germinated on coco peat based substrate were transferred to earthen pots (size: 26cm height, 26 cm diameter). Pot mix consist of red soil, sand and organic compost in 1:1:2 ratio, plants were grown in the green house [average temperature 28°C/20°C (day/night), RH (60-65%)] till anthesis stage. Third instar larvae of S. litura were starved for 12 hours before herbivore induction in experimental plants. Two larvae per plant were allowed on third leaf from top, to account a damage area of 5mm 2 /per larva and were removed thereafter. Top three leaf samples were harvested post 4hrs of herbivory, frozen in liquid nitrogen and stored at -80°C until use. RNA extraction and PCR conditions . Total RNA was isolated from 1g of leaf samples of all three tomato cultivars (uninduced and herbivory induced), using standard phenol extraction method (Cheng and Team 1998). RNA was quantified using Cary 50 UV-Vis spectrophotometer, (Varian Australia Pty Ltd, Mulgrave, Vic, Australia) at A260nm and qualitatively by A260/A280nm ratio (>1.8). The integrity of RNA was confirmed by electrophoresis on 1% agarose gel. cDNA was synthesized at 37°C for 30min, using 5 μg RNA in the presence of 4 U of M-MLV reverse transcriptase (USB corporation, Cleveland, Ohio, USA) after digesting with 2U DNase 1 (Invitrogen, Mexico, USA) following manufacturer’s instructions. Gene specific primers (Table 1) designed for four target genes COI-1, DBP, LAP and PPI were used for PCR post cDNA synthesis. The cDNA was synthesized by priming with oligo dT primers. Plant Actin was optimized as reference transcript for cDNA synthesis and PCR amplification. RT-PCR was performed using 8 µl of cDNA template, 0.2 mM each of forward and reverse gene specific primer, 0.2 mM dNTPs, 1 U of FideliTaq DNA polymerase(USB corporation, Cleveland, Ohio, USA) and 1 x PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl 2 , pH 8.6) in a final volume of 25 µl. PCR was carried out on a DNA Engine, Peltier Thermal Cycler (BIO-RAD, Rock way, NJ, US) using the following cycling conditions: initial denaturation at 94 °C for 3 min, followed by amplification cycles at 94 °C for 45 s, 50 °C for 45 s, and 72 °C for 1 min and then a final extension at 72 °C for 10 min. The PCR was done at two different cycle lengths for quantification of the transcripts. No enzyme (Reverse transcriptase) controls for each RNA sample were performed parallel to know genomic DNA contamination. PCR product were resolved using electrophoresis in 1.2% agarose gel stained with ethidium bromide (EtBr), documented under UV light on UVIpro platinum gel documentation system. All amplicons were sequenced to confirm the target gene specificity of designed primers (data not shown). Densitometric analysis of EtBr stained gel bands was performed on UVIBandMap image analysis software (Eurodyne Ltd, Lindale, Cumbria, UK). Statistical analysis. Data of target genes band intensity value were normalized to reference (Actin) gene band intensity of similar cDNA samples. Mean and standard error of all samples were calculated after normalization to reference gene. A pairwise comparative Tukey’s (HSD) test was performed using XLSTAT software (Addinsoft, Damrémont, Paris, France). RESULTS AND DISCUSSION Gene expression levels in uninduced state of plants shows variance across three cultivars. Performing pairwise comparative Tukey’s (HSD) test on mean o f uninduced expression shows that difference across was not significant. COI-1 and LAP expression levels are high in comparison with the other two transcripts in tomato cultivar.