| The research of transgenic technology has been highly concerned at home and abroad,but the safety of screening marker genes has been a topic of discussion,the removal of marker genes is an important measure to solve the safety problems of genetically modified.(E)-?-farnesene is a kind of sesquiterpene hydrocarbons.The sesquiterpene(E)-?-farnesene is the alarm pheromone for many species of aphids,which can be applied to disperse aphids in plants.(E)-?-farnesene has important role on environmentally friendly,pollution-free,green environmental protection and the protection of natural enemy.In this study,the(E)-?-farnesene synthase gene has been transformed into Arabidopsis thaliana mediated by Agrobacterium tumefaciens.The results are indicated as following:(1)The basal vector p ART7-E?F containing the target gene E?F was modified to p NMCS-E?F,and then using the excessive carrier p NMCS-E?F and containing the self deleted structure Cre/loxp of the bare vector PNCX was constructed with the self delete vector PNCX-E?F which contained the target gene and the selected marker gene of the neomycin phosphotransferase gene(npt II).(2)The vector PNCX-E?F was transformed into Arabidopsis thaliana mediated by Agrobacterium tumefaciens.The antibiotic G418 was selected as screening agent.The optimal concentration of G418 was determined,the value was 20 mg/L in the culture medium and was 50 mg/L in the soil.(3)PCR technology and G418 were used to screen positive lines of Arabidopsis thaliana.The single copy lines which accord with Mendel's Inheritance Law of 3:1 were obtained.(4)The transgenic lines and the wild type were used to test the effectiveness of aphid repelling.The results showed that aphid number of transgenic lines was significantly lower than that of the wild type,and the aphid repelling effect is obvious.(5)T2 generation positive homozygous lines were treated with ?-estradiol to induce selection marker gene self deleted.We found that add ?-estradiol into the c?Lture medium,the optimum concentration is 100 ?mol / L;the optimal concentration is 20 ?mol / L for hydroponic methods.(6)Screening of ?-estradiol treated T3 generation of the transgenic lines by G418,take the loss of resistance plants as marker deleted plants,found Cre/loxp self deleted and efficiency can reach 65%.(7)The first and second lines' T3 and T4 generation seedings were treated with ?-estradiol that were detected by PCR.The results showed that the selected marker genes(npt II)were deleted in the seedlings of T3 and T4,and the removal efficiencies were 11%-34.3%.Moreover,experimental results also get the generation higher as well as the self deleted efficiency higher,and the self deleted efficiency of the possibly for a single copy of the first line is higher than of the possible for a multiple copy of the second line.This study shows that it is feasible to use the self deleted system to carry out the self deletion of the marker gene,which lays the foundation for obtaining the aphid-resistance transgenic lines with no selectable marker. |
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