Construction of plant expression vectors harboring WRI۱ gene and optimization its transformation in tobacco plants

Background and Aim: Increasing the yield of oilseed crops is an important goal of plant breeding. Several genes involved in triacylglycerol metabolism have previously been reported to enhance the oil content of seeds when their expression is altered. In this study, to increase the amount of oil, the WRI۱ gene expression construct was designed and constructed, which is one of the effective genes in the oil production pathway. To construct, the WRI۱ gene fragment was separated by specific restriction enzymes from the PGH.WRI۱ cloning vector and cloned into the PGH.O۳.۲.۲ intermediate vector under the SBP promoter and E۹ terminator. The WRI۱ gene cassette was then inserted into the pBin۱۹ binary expression vector. The resulting construct was transferred to Agrobacterium tumefacience strain EHA۱۰۵. To verify the structure and expression of the gene fragment, a transgenic tobacco model was used. Molecular evaluation of transgenic plants confirmed the presence and activity of the WRI۱ gene. Seeds from transgenic plants in the next generation in the medium containing kanamycin produced strong and healthy seedlings.Methods: WRI۱ Gene synthesis and vectors: The WRI۱ gene cDNA sequence (Arabidopsis origin) with accession number of KM۰۸۵۴۴۸.۱ was obtained from NCBI database, in the next step as using preferred codons have a positive effect on enhancing gene expression according to the genomic sequence of safflower sequences codon was optimized by codon optimization software. Also, cleavage sites of XhoI and AvrII enzymes were added to the gene sequence in order to cloning to the PGH.O۳.۲.۲ vector. The intermediate construct PGH.O۳.۲.۲ with SBP specific seed promoter and E۹ terminator and pBin۱۹ expression construct with the LacZ gene and kanamycin selective marker were used (Figure ۱). WRI۱ gene sequences were synthesized by Neda Fan Company. The required enzymes and solutions were obtained from Cinnagen and Fermentaz co. Preparation of susceptible bacteria, digestion reaction, ligation reaction, product transfer to susceptible bacteria, and plasmid DNA extraction was performed according to the instructions of Russel & Sambrook (۲۰۰۱) (۶). Gel purification was performed using the Roche High Pure PCR Purification kit. Preparation of WRI۱ monogenic construct: By using heat-shock protocol (Green & Sambrook ۲۰۱۲) (۲) PGH.WRI۱ Plasmid was cloned into the XL۱blue cells of E. coli using the XhoI and AvrII cleavage sites. The WRI۱ gene was digested from the PGH vector and then cloned in the PGH.O۳.۲.۲ intermediate vector, PGH.O۳.۲.۲ digested with SalI and SpeI enzymes. The presence of the gene in this vector was confirmed by enzymatic digestion (Fig ۲A). The WRI۱ gene cassette was cleaved by HindIII restriction enzyme and insert in the digested pBin۱۹ expression vector. The pBin۱۹.WRI۱ construct was confirmed by enzymatic digestion and PCR test by SBP primers with F: AAACTCTGATTGAAC CTAC and R: GGATTCTTTGTGTTGTAC sequences (Fig. ۲ b and c). The resulting recombinant construct was then transferred to Agrobacterium strain EHA۱۰۵ (containing Rifampicin ۷۵ mg/l antibiotic-resistant construct), the resulting construct was used in other stages of gene transformation. Agrobacterium Infection and Co-cultivation A single Agrobacterium colony derived from Agrobacterium cultured in ۲۰ ml liquid LB medium containing ۱۰۰ mg/L kanamycin and ۷۵ mg/L rifampicin on a shaker (۱۸۵ rpm) at ۲۸C for ۱۶ hrs. Explants were transferred to the bacterial suspension and shacked gently for about ۱۵ min and then blotted on a sterile filter paper for ۵ min. Infected explants transferred to the Co-cultivation medium under dark conditions for ۲ days at ۲۸°C. After Co-cultivation, the explants washed with sterilized distilled water, then explants blotted on sterile paper for ۱ minute then dried explants transferred to selection medium (MS + ۱۰۰mg/L kan + ۲۵۰mg/L Cefotaxime + ۲mg/L BAP + ۰.۱mg/L NAA) for ۲–۳ weeks and they were subcultured onto fresh medium every۱۵ days. Healthy and elongated shoots transferred to the rooting media (MS+ ۱۰۰mg/L kan + ۲۵۰mg/L Cefotaxime + ۱mg/L NAA) for ۲ weeks. Rooted healthy plants transferred to pots filled with vermiculite (۲۰%), perlite (۴۰%), and peat (۴۰%) and later to the greenhouse. Molecular characterization of putative transgenic plants Genomic DNA from young leaves of putative transgenic plants was extracted using CTAB protocol (۸). The presence of the WRI۱ gene was confirmed by specific SBP primers and the internal actin gene. The recombinant pBin۱۹ plasmid was used as positive control and non-transgenic plants and DNA-free PCR reaction material were as a negative control. The reaction was performed at ۹۴ ° C for ۴ minutes and ۳۵ cycles (each cycle consisting of ۹۴° C for one minute, ۶۵° C for ۳۰ seconds, ۷۲° C for ۴۰ seconds and completion of expansion for ۳ minutes). Vir primers were also used to check Agrobacterium contamination.Results: Results The accuracy of WRI۱ gene isolation and its insertion in the intermediate PGH.O۳.۲.۲ construct was confirmed by HindIII enzymatic digestion, and observation of ۳۷۷۰ and ۲۹۱۱ bp bands, respectively (Fig. ۲ a). The Integrity of pBin۱۹.WRI۱ expression vector was confirmed by PCR assay using SBP-specific promoter primers (Fig. ۲ b) as well as HindIII enzymatic digestion pattern (Fig. ۲ c). The pBin۱۹.WRI۱ construct was transferred to Agrobacterium strain EHA۱۰۵ and, after transformation confirmation, recombinant bacteria were used in gene transformation procedure. Plants that survived in the kanamycin-selective medium for two months were considered as probably transgenic events after and more confirmed by PCR assay using specific primers SBP (۵۷۴ bp) and internal actin gene primer (۲۰۰ bp). Vir primers and PCR tests were also used to determine the non-contamination of Agrobacterium specimens (Fig. ۲ d and e). Plants that were SBP-positive PCR and Vir-negative were selected as transgenic plants and transferred to pots. Seeds from probably transgenic plants were sown in culture medium containing ۱۰۰ mg/L kanamycin. After about a month, seeds from transgenic plants, produced green and healthy seedlings, but seedlings from control seed were weak and yellowish (Fig. ۳). PCR with the SBP primers and the actin internal gene primer and the presence of ۵۷۴ of and ۲۰۰ bp fragments respectively, indicate the presence of SBP promoter and confirmed WRI gene insertion in the transgenic plant (Fig. ۲ e).Conclusion: Seeds from transgenic plants in the next generation in the medium containing kanamycin produced strong and healthy seedlings.

کلیدواژه ها:

Agrobacterium tumefacience ، Seed oil ، tobacco ، WRI۱