| The ethanol inducible system consists of two parts, respectively AlcR protein and an inducible promoter, in the presence of ethanol, it can change the AlcR protein conformation, making the original activity of the protein was activated, the activated protein identifies inducible promoters, such as the AlcR specific recognition sites on alcA promoter, makes the promoter activated and the target gene expressed.The ethanol inducible system which derived from Aspergillus nidulans can control the expression of target gene in time and space. The ethanol inducible system in the use of ethanol-induced marker gene deletion verification tests found that the ethanol-induced system in prokaryotes expression constitutively. In this study, we took the Aspergillus nidulans genome as a template, cloned the natural promoter alcS, alcM, alcP, alcU, alcR, alcA and aldA which regulated by AlcR. Using the Cre/loxP bit site specific recombination system which Cre protein can identify the two loxP bit points and deletion or restructuring. Verification in the prokaryote E.coli, expression of natural promoter start the sub Cre protein in the non-induced conditions. Specific experimental results were:(l) alcS in prokaryotes, started the protein Cre expression in non-induced conditions and delete resistance gene fragment between the two same direction loxP bit of pYBA100;(2) alcA, alcU and alcR in prokaryotes, started the protein Cre expression in non-induced conditions, and resistant fragment between the two same direction loxP bit recombinated in pYBA100;(3) alcP,alcS and aldA in prokaryotes, did not start the expression of protein Cre in non-induced conditions, the deletion of resistant fragment between the two same direction loxP bit in pYBA100did not happen.The experimental results above show that, in the non-induced conditions, natural promoters alcP,alcS and aldA is inducible promoter, the alcA,alcU, alcS and alcR is constitutive promoter.With the arrival of the genetically modified, herbicide resistance of transgenic crops has large scale cultivation, glyphosate-resistant transgenic crops as the one has been widespread concern, and the growing number of glyphosate resistance gene. Registered patents on glyphosate-resistant EPSP synthase coding sequence found that the EPSPS gene derived from Agrobacterium tumefaciens C58was identified of resistance prokaryotes, not verify resistance in plants.In this study, Agrobacterium tumefaciens C58as a template amplification the EPSPS and transformation and build a binary expression vector, then Agrobacterium-mediated transformed into Arabidopsis. Measurement of resistance by glyphosate concentration for screening of transgenic seedlings and the experimental results were:In genetically modified blade coating applied glyphosate concentration of0.02%, the transgenic positive seedlings yellow withered, low resistance performance. Based on this summary of15patents which are about glyphosate-resistant genes in recent years, we made a gene phylogenetic tree analysis by glyphosate amino acid sequencing. The experimental result were:(1) EPSPS resistance gene from Agrobacterium tumefaciens C58in glyphosate-tolerant plants was very low, have no practical value;(2) Made a gene phylogenetic tree analysis by glyphosate amino acid sequencing which are already known found that these EPSPS could be divided into two categories by whether or not containing the conserved sequence LGNAGTA;(3) I type (except patent9[7]) contained LGNAGTA conserved sequences which were not found in type Ⅱ;(4)The EPSPS in plant belongs to I type.The new ethanol inducible system can be used for exogenous gene expression, and regulation of target gene expression in time and space. Different sources of glyphosate-resistant EPSPS gene can also be induced by ethanol system and regulation of expression. Inducible system and resistance genes can build a carrier and transferred to the plants, achieve the controllability of the resistance gene. |
PDF Full Text Request