The Construction Of Plant Expression Vector By Gateway Technology And Studies On Selection Markers Of Plant

GatewayTM recombinant technology was developed by Invitrog-en of USA,which was a rising new construction method of cloning vector in recent years. In this study, in order to construct the high-throughput plant expression vector, the pYBA backbone has been transformed and the streptomycin resistance genes has been replaced the original bacteria selection marker kanamycin resistance gene, by the method of digestion and connection, digested plant selectable marker expression cassette (Npt, Hyg and Bar),35s-Pro,35stop polyA, fluorescent protein (EGFP, EYFP, ECFP, mVenus, mTFP1, TagRFP and mCherry) attR sites were connected in sequence. Gateway target carrier with seven kinds of fluorescent protein tags were successfully been constructed. Target gene LST8.2and PGK.1were amplification by PCR,then integrate these two genes into the entry vector pENTR3c.Based on Gateway LR recombination reaction, the target gene of LST8.2and PGK.1has been Successfully reorganized in Recombinant vector of TagRFP and Cherry with labels.The optimal selection marker gene played a multiplier effect for genetic transformation of plants during construction of vector. In this study, we selected cp4-epsps, gat4621, GR79-epsps and gatm gene as resistant comparison.It integrated these four resistance genes respectively and disseminated Arabidopsis through pYBA(?) plant expression binary vector.The results showed that:(1)Four kinds of glyphosate-resistant gene survival rate in descending order is gat4621> gatm> CP4-epsps> GR79-epsps.(2)Transgenic Arabidopsis seedlings of CP4-epsps and GR79-epsps present some symptoms such as yellowing leaves and high mortality at low concentration, gat4621and gatm growing well, the survival rate is high.(3) CP4-epsps and GR79-epsps remaining high resistance seedlings and growing well at high concentrations, transgenic seedling of gat4621survival rate is still high, most of gatm and gat4621appear bolting obstacles.(4) CP4-epsps and GR79-epsps of TO generation seed above0.004concentrations are seedless fruit, transgenic plants of EPSPS of over expressing could lead accumulation of glyphosate in plants easily, so that the plant appeared adverse traits such as abortion, decreased rates of pollination. Stop spraying glyphosate to gatm and gat4621,Arabidopsis plants showed following features such as bolting recovery.(5)The total DNA were extracted from transgenic Arabidopsis, glyphosate resistance genes has all integrated into plants through identification.(6)Four kinds of resistance genes in highly resistant materials and weak anti-expression have significant differences by quantitative PCR comparison,gatm and gat4621is more suitable for transgenic plant selection marker, can filter out transgenic seedlings of low concentrations effectively, has no effect on plant growth and development, improve screening efficiencyIn order to find a safe plant selectable marker gene, in this study, dsdA gene from Escherichia coli was investigated. dsdA can catalyze D-serine into keto acid, ammonia and water, is not toxic for the transgenosis plant, but also metabolites of plants provide carbon and nitrogen. This article cloning dsdA genes in E. coli, and import it into pYBA1305expression vector, verify dsdA resistance genes, the results showedthat it is obvious that the transgenic plants has resistance, dsdA can be applied to plants as a selectable marker.