《Table 1.Nucleotide sequences used in construction of fusion vector with chloroplast transit peptide

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《Functional Analysis of Three Rice Chloroplast Transit Peptides》

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To construct the p35S::CTPTRXz-GFP vector，we generated a DNA fragment encoding the N-terminal56 amino acids of TRXz by PCR using p35S::CTPTRXz-GFP primer（Table 1）from the NPB cDNA.The PCR procedure was as follows:Denaturation at94oC for 4 min，followed by 32 cycles of 98oC for 10s，annealing at 55oC for 30 s，extension at 68oC for30 s，and a final extension step at 68oC for 10 min.The PCR products were analyzed on 2%agarose gels.We used the Wizard SV Gel and PCR Clean-Up System（Promega Biotech，USA）to extract and purify the target DNA fragment.The purified DNA fragment encoding the N-terminal 56 amino acids of TRXz was fused with the purified p35S::GFP fragment using a ClonExpress II One Step Cloning Kit（Vazyme Biotech，Nanjing，China）by homologous recombination.The ligation reaction was conducted for 30 min at37oC in a 20μL reaction，consisting of 2μL PCR product，2μL purified p35S::GFP fragment，4μL of5×CEII buffer，2μL Exnase II and 10μL sterile water.After transformation into Escherichia coli（DH5α），the positive clones were identified by PCR using the s65t-1F（GAGGACAGGCTTCTTGAG）and s65tR（GGTGGTGCAGATGAACTT）primers and sent to Hangzhou Tsingke Biotech Company（Hangzhou，China）for sequencing verification using the s65t-1F and s65tR primers.The other CTP and GFP fusion protein vectors（p35S::CTPHSA1-GFP and p35S::36CTPFLN1-GFP）were constructed and verified as described above using primers listed in Table 1.