The addition amount S for both r and t were set

September 19, 2017

The addition amount S for both r and t were set equal to the estimated N0 of r. Therefore, Sr is equal to St, and consequently, transgene copy number sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (3-2Rt )2 Rt 3-2Rt z { 2 1-Rt 2|(1-Rt ) 4|(1-Rt ) Hesperidin chromosome ploidy| sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (3-2Rr )2 Rr 3-2Rr z { 4|(1-Rr )2 1-Rr 2|(1-Rr )Fb (N0 zS)|(1zE) (N0 z3S) Fc?6??0?”>(16)Then the following equation can be obtained from Equation (15) divided by Equation (16). Fa |Fc N0 |(N0 z3S) Fb 2 (N0 zS)2 ?7?Transgenic Tomato PlantsTransgenic tomato (Solanum lycopersicum Linn. cv. Ailsa Craig) plants, carrying an integrated Escherichia coli hygromycin phosphotransferase gene (HPT), were previously obtained via Agrobacteriummediated transformation. Primary transformants (T0) lines and wild type plants were used in this study.To simplify the formula, set R = (Fa6Fc)/Fb2, then the following equation can be derived. R N0 |(N0 z3S) (N0 zS)2 ?8?Genomic DNA ExtractionYoung leaves were collected and ground to a fine powder in liquid nitrogen. Approximately 100 mg of leaf tissue was used to isolate DNA for PCR with a DNeasy Plant Mini kit (Qiagen), according to the manufacturer’s instructions. Approximately 1 g ofIn Equation (18), N0 is the only unknown factor and can be calculated as follows:A qPCR Approach for Transgene Copy Number AnalysisTable 1. Primers used in this study.Gene ELIPGenBank ID AYForward primer (59?9) GGTTCGCGATCTAGACAATACTaReverse primer (59?9) CAAAATGAAAAGCTTTATATACTC AACACGCGAAGTCCTATGAA CAGCACTCGTCCGAGGGCAAAGG GGCGTCGGTTTCCACTAT TTGGCGACCTCGTATTGGb bApplication cloning qPCR cloning qPCR probe labelingPCR product size (bp) 1019 200 1013 200ACAATACTAGTACTTCTTCACCTTT HPT V01499 GCCTGAACTCACCGCGACGTCTG GCTCCGCATTGGTCTTGA GATCGTTATGTTTATCGGCACTa The underlined nucleotides are the restriction site for Xba I; doi:10.1371/journal.pone.0053489.tThe underlined nucleotides are the restriction site for Hind III.leaf tissue was used to isolate DNA for Southern blot following an improved CTAB method [27].Primer DesignTomato early light-induced protein (ELIP) gene, a single-copy gene [28] was selected as the internal reference gene (r), and E. coli HPT as the integrated target gene (t) in this study. Primer pairs were designed for gene cloning or qPCR, using Primer Premier 5 (PREMIER Biosoft International) according to sequences deposited in GenBank (Table 1).enzymes exists in the plant expression vector used for transformation and no site was present in the hybridization probe. The digested DNA was separated on 0.8 agarose gel, and then transferred to a positively charged nylon membrane (Roche) according to Sambrook and Russell [29]. DIG-labelled HPT probe DNA was prepared by PCR with the primers listed (Table 1), hybridization and autoradiography were performed according to DIG Application Manual for Filter Hybridization (Roche).Results and Discussion Recombinant Plasmid ConstructionELIP and HPT were amplified by PCR and then ligated into pUCm-T (TaKaRa) to generate plasmids pELIP and pHPT according to traditional protocols of Sambrook and Russell [29]. Following simultaneous digestion of the plasmids with Xba I and Hind III, the 995-bp 301353-96-8 fragment from pELIP digestion and the 3768bp fragment from pHPT digestion were recovered and ligated to generate recombinant plasmid pHE (Figure 2).The addition amount S for both r and t were set equal to the estimated N0 of r. Therefore, Sr is equal to St, and consequently, transgene copy number sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (3-2Rt )2 Rt 3-2Rt z { 2 1-Rt 2|(1-Rt ) 4|(1-Rt ) chromosome ploidy| sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (3-2Rr )2 Rr 3-2Rr z { 4|(1-Rr )2 1-Rr 2|(1-Rr )Fb (N0 zS)|(1zE) (N0 z3S) Fc?6??0?”>(16)Then the following equation can be obtained from Equation (15) divided by Equation (16). Fa |Fc N0 |(N0 z3S) Fb 2 (N0 zS)2 ?7?Transgenic Tomato PlantsTransgenic tomato (Solanum lycopersicum Linn. cv. Ailsa Craig) plants, carrying an integrated Escherichia coli hygromycin phosphotransferase gene (HPT), were previously obtained via Agrobacteriummediated transformation. Primary transformants (T0) lines and wild type plants were used in this study.To simplify the formula, set R = (Fa6Fc)/Fb2, then the following equation can be derived. R N0 |(N0 z3S) (N0 zS)2 ?8?Genomic DNA ExtractionYoung leaves were collected and ground to a fine powder in liquid nitrogen. Approximately 100 mg of leaf tissue was used to isolate DNA for PCR with a DNeasy Plant Mini kit (Qiagen), according to the manufacturer’s instructions. Approximately 1 g ofIn Equation (18), N0 is the only unknown factor and can be calculated as follows:A qPCR Approach for Transgene Copy Number AnalysisTable 1. Primers used in this study.Gene ELIPGenBank ID AYForward primer (59?9) GGTTCGCGATCTAGACAATACTaReverse primer (59?9) CAAAATGAAAAGCTTTATATACTC AACACGCGAAGTCCTATGAA CAGCACTCGTCCGAGGGCAAAGG GGCGTCGGTTTCCACTAT TTGGCGACCTCGTATTGGb bApplication cloning qPCR cloning qPCR probe labelingPCR product size (bp) 1019 200 1013 200ACAATACTAGTACTTCTTCACCTTT HPT V01499 GCCTGAACTCACCGCGACGTCTG GCTCCGCATTGGTCTTGA GATCGTTATGTTTATCGGCACTa The underlined nucleotides are the restriction site for Xba I; doi:10.1371/journal.pone.0053489.tThe underlined nucleotides are the restriction site for Hind III.leaf tissue was used to isolate DNA for Southern blot following an improved CTAB method [27].Primer DesignTomato early light-induced protein (ELIP) gene, a single-copy gene [28] was selected as the internal reference gene (r), and E. coli HPT as the integrated target gene (t) in this study. Primer pairs were designed for gene cloning or qPCR, using Primer Premier 5 (PREMIER Biosoft International) according to sequences deposited in GenBank (Table 1).enzymes exists in the plant expression vector used for transformation and no site was present in the hybridization probe. The digested DNA was separated on 0.8 agarose gel, and then transferred to a positively charged nylon membrane (Roche) according to Sambrook and Russell [29]. DIG-labelled HPT probe DNA was prepared by PCR with the primers listed (Table 1), hybridization and autoradiography were performed according to DIG Application Manual for Filter Hybridization (Roche).Results and Discussion Recombinant Plasmid ConstructionELIP and HPT were amplified by PCR and then ligated into pUCm-T (TaKaRa) to generate plasmids pELIP and pHPT according to traditional protocols of Sambrook and Russell [29]. Following simultaneous digestion of the plasmids with Xba I and Hind III, the 995-bp fragment from pELIP digestion and the 3768bp fragment from pHPT digestion were recovered and ligated to generate recombinant plasmid pHE (Figure 2).