In-silico characterization of promoters and asal gene
The cis-regulatory elements within the 2X35S constitutive promoter and rolC phloem specific promoter were identified using PlantCARE software for comparative analysis (Lescot et al. 2002).
The nucleotide sequence of ASAL was translated through Swiss Bioinformatics Server (Artimo et al. 2012). Signal peptide and subcellular localization of protein was predicted by TargetP 1.1 Server (Emanuelsson et al. 2000). Molecular weight and isoelectric point of protein was computed using in-silico analysis (Gasteiger et al. 2005). Domain was predicted by online PROSITE scan tool (Sigrist et al. 2012). Protein structure was predicted by I-TASSER (Roy et al. 2010; Yang et al. 2015). Protein model having C-score =-2.99, Estimated TM-score 0.38±0.13 and Estimated RMSD = 12.0±4.4Å was selected. Protein Model predicted by I-TASSER was submitted to ConSurf server (Ashkenazy et al. 2010; Ashkenazy et al. 2016) for prediction of evolutionary conserved residues. Phylogenetic analysis of ASAL was conducted by MEGA 6.0 (Maximum Likelihood) while Motif prediction in all the orthologues of ASAL was identified by Motif finder.
Bulblets of Allium sativum (garlic) were obtained from Ayub Agricultural Research Institute (AARI), Faisalabad, Pakistan and grew them in peat moss under control conditions at 27-28 °C and 54-67% relative humidity. Seeds of Nicotiana tabacum were sterilized with mixture of 20% chlorox plus, 0.02% Tween 20, rinsed with distilled water and dried to sow on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) under aseptic conditions at 27-28 °C and 54-67% relative humidity.
Construct development (35S-ASAL-CaMV and rolC-ASAL-CaMV)
Total RNA was isolated from garlic using Plant RNA Purification Kit (Cat No.12322-012, Invitrogen) to synthesis cDNA using H-Minus First Strand cDNA Synthesis Kit (Cat No. K1632, Thermo Fisher) according to the manufacturer’s protocol. Primers were designed in-silico using Primer3 program (Untergasser et al. 2012) to amplify full length asal gene from garlic. RT-PCR of 50 µl reaction mixture was conducted using proofreading DNA polymerase, pfu (2.5 U), and cDNA (1 µg) to amplify 546 bp full length asal gene using 10 µM of gene-specific primer pair, ASAL-F3 and ASAL-R3 (Table 1). Reaction mixture was run in thermal cycler under following conditions; initial denaturation at 94° C for 5 min followed by 39 cycles of 94 °C for 1 min, 63°C for 1 min, 72°C for 1 min and final extension of 72 °C for 5 min. Amplified gene was eluted using a Gel Extraction Kit (Cat No. K2100-12, Invitrogen) and cloned in PCR blunt cloning vector (Cat No. K2700-20, Invitrogen). Resulting clone was confirmed through restriction analysis and Sanger sequencing.
Full length asal gene was restricted from PCR blunt vector using HindIII and SmaI and cloned in pJIT163 having 2X35S promoter and CaMV terminator. Gene cassette, 35S-ASAL-CaMV was restricted from resulting plasmid pIT163-ASAL using SacI and EcoRV and cloned in plant transformation vector, pGA482. The resulting plasmid, pGA482-35S-ASAL was confirmed through restriction analysis.
2X35S promoter of pIJT163 was replaced with rolC promoter to clone full length asal gene under phloem specific rolC promoter. Engineered gene cassette (rolC-ASAL-CaMV) was cloned in pGA482. The resulting plasmid, pGA482-rolC-ASAL was confirmed through restriction analysis.
Agrobacterium mediated tobacco transformation
Plasmids pGA482-35S-ASAL and pGA482-rolC-ASAL were separately, electroporated in Agrobacterium tumefaciens strain, LBA4404 and their cultures were developed. For stable transformation, leaf discs of Nicotiana tabacum (cv. Samsun) (Amaya 1997) were co-cultivated with engineered cultures of A. tumefaciens. and kept over the regeneration media containing MS salt and sucrose to develop transgenic tobacco plants..
Molecular analysis of tobacco plants
All putative transgenic tobacco plants were screened using gene specific and construct specific primer pairs (Table 1). CTAB (cetyl trimethylammonium bromide) method (Doyle and Doyle 1987) was used to isolate genomic DNA from leaves of tobacco plants expressing ASAL under 35S and rolC promoters, respectively. PCR reaction mixture of 25 µl was prepared using 100 ng of DNA, 12.5 µl of Dream Taq Green PCR Master Mix (2X) (Cat No. K1081, Thermo Scientific), and 1 µl of each primer (10 µM). Gene specific primers were used to confirm the presences of asal gene while construct specific primers were used to detect the promoter-gene fragment (35S-ASAL or rolC-ASAL) in transgenic tobacco plants. PCR reaction mixture was run in thermal cycler at 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 63°C for 1 min, 72°C for 1 min and final extension of 72°C for 5 min. The PCR amplified products were checked on 1% agarose gel to screen transgenic tobacco plants.
Integration and copy number of transgene (asal) was detected in T0 tobacco lines through Southern blotting. For this genomic DNA was extracted from the transgenic tobacco expressing ASAL under 35S and rolC promoters. DNA (100 µg) samples were restricted using EcoRI at 37°C overnight and then subjected to the electrophoresis using 0.8% agarose gel at 25 volts overnight. Fractionated DNA fragments were transferred to the positively charged nylon membrane. Nylon membrane was crosslinked with UV linker (0.240 J/cm2) (Strata linker) and hybridized with DIG (digoxigenin) labelled probe. Nylon membrane was washed using Dig Wash and Blocking Buffer Set (Cat No.11585762001 Roche) and signals were developed using color substrate NBT/BCIP.
qRT-PCR for gene expression analysis
qRT-PCR was conducted for relative gene expression analysis of asal gene in T0 transgenic tobacco lines. RNA was isolated from the 3-4 leaf stage (45 days old) transgenic and non-transgenic tobacco plants using SV Total RNA Isolation System (Cat No. Z3101, Promega). Primers were designed using Primer3 program. Concentration of primers were optimized, and qPCR reaction mixture was prepared containing, cDNA (200 ng), Power SYBR Green PCR Master Mix (Thermo Scientific), gene specific primers (qASAL-F5, qASAL-R5) and 18S rRNA as internal control (Table 1). Three replicates/sample were loaded on 96 well plate to run in Quantstudio 6 Real-time PCR system (Thermo Fisher Scientific) using following conditions; 95 oC for 3 min, 39 cycles at 95 oC for 30 sec, 56 oC for 30 sec and 72 oC for 50 sec. Relative gene expression of each transgenic tobacco line was measured with ΔΔCt method.
A culture of a tobacco-adapted M. persicae strain (Ramsey et al. 2007; Ramsey and Jander 2008) leaves of tobacco lines, LS-15, LS-17, LS-18, LS-20, LS-21 and LS-25 expressing ASAL under 2X35S promoter and tobacco lines, LR-1, LR-3, LR-7, LR-10, and LR-12 expressing ASAL under the rolC promoter were placed on Petri plates containing 1% agar. Detached leaves of non-transgenic tobacco were set parallel to transgenic tobacco lines as control. Five adult aphids were released on each leaf of transgenic and non-transgenic plants. Mortality and fecundity of adult aphids were observed after every 24 hours for thirteen days.
Significant difference of relative gene expression was determined between transgenic and non-transgenic tobacco plants using ANOVA followed by LSD. Mann-Whitney U-tests was used to calculate significant difference of mortality and fecundity between aphid feeding on transgenic and non-transgenic tobacco.