Market-free Transgenic System Establishment Of Citrus And Preliminary Application

The selectable marker genes are required to ensure the efficient plant genetic transformation, The presence of antibiotic or herbicide resistant selective marker genes in transgenic plants causes the concerns of the potential hazards on ecological environment and human health. Among the marker gene elimination strategies, inducible auto-excision presents the most promising choice.Citrus canker is a disease of world-wide quarantine and have done great harm to the industry. Due to lack of resistant resources, effective disease prevention and control methods, the disease still unable to eradicate. pthA-NLS have the function of pathogenic agents, One of the main purposes of this thesis was to construct a chemical-inducible marker-free system for citrus and eliminate the selectable marker gene from pthA-NLS transgenic citrus. The results of this study are as follows:1.In this report, we developed the research of marker-free transgenic'Succari' sweet orange by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination in a single transformation. The feasibility that this chemical-inducible excising marker gene system was applied on citrus was proved and an efficient marker-free transgenic system in citrus was established.the express of gfp could be observed successfully by fluorescence microscope. Finally, Molecular detection indicated that the completed DNA recombination and excision in transgenic citrus were alreadly occurred after 30 days grafted in vitro.2.The plasmid of 'p35S-NLS'was constructed in through the exchange from gfp to pthA-NLS.3.pthA-NLS was transfered to Dahong sweet orange by Agrobacterium-m ediated.27 transgenic strains were obtained through PCR. There no recombinati on in transgenic plants by multiplex PCR using P1 P2 and P4.Then them were detected through RT-PCR after 30d during continue induction (6μMβ-estradi ol),recombination was detected.