The Key Technology Of Foreign Gene Safety Transformed System In Plants

The biosafety of transgenic plants was always a key problem in the world. We transformated the tranditional plant expressed vector by biotechnology, to obtain transgenic product without selectable marker gene, target gene and target peotein.The DNA fragment of chloroacetanilide-induced promoter (In5-2) was amplified by PCR method from maize genome and fused with GUS gene to construct the expression vector pCGUS for tobacco transformation. The results showed that the GUS gene was expressed under chemical induction in different adjuvant concentration and inducing time, in which the GUS activity increased correlatively with the chemical concentration and decreased when the inducing time was elongated. Maize In5-2 promoter was induced by herbicide-safener. The boundaries required for maximal promoter expression were defined by 5' deletion analysis of the maize In5-2 promoter fragments coupled to a GUS reporter gene. The expression patterns of these chimeric gene constructs were evaluated in transgenic Nicotiana tabacum. Analysis of the 5' deletion constructs showed the regulation region of its basic transcription was mainly within - 1520 to - 1431 bp upstream of ATG Two stem-loop structures were within - 1108 to - 1079 and - 891 to - 864 bp relative to ATG It suggests that their protein binding site is between - 1079 to - 897 bp relative to ATG. In addition, cis-acting elements responsive to chloroacetanilide could be mainly located within the range of - 388 to - 86 bp upstream of ATGA new inducible Cre/lox system was constructed in transgenic tobacco plants. The inducer-treatment of tobacco callus mediates an excision event in which the selectable marker gene and Cre gene between two lox sites were deleted. A chloroacetanilide-induced promoter (In5-2) was used to control the expression of Cre gene in this system. Molecular analysis of transgenic tobacco plants showed the interest gene, gus, was integrated into the genome whether removing has been successful, and forty-five out of forty-eight T0 plants were transgenic tobacco without the marker gene, hpt. This system uses a single vector to circumvent the flaw of other dual recombinase vector systems.A new transgenic plant product that has not marker gene, target gene and target protein was obtained. It is derived from a 3-free system, the novel system included R/RS marker-free system, green tissue- specific expression system and chemical-inducible Cre/lox target gene excision system. The system was tested with the transgenic CrylAc tobacco. And the results showed that the selectable marker gene was deleted in medium culture, the target gene was moved from plant genome after induced by spraying of herbicide safener, the target protein in the leaf and root was degraded. This system could provide a new way to resolve the problem of biosafety in transgenic plants.The non-transgenic nprA (neutral protease A) gene was amplified by PCR, and it was modified according to plant code bias. The modified nprA gene was artificially synthesized, and the synthesized nprA gene was renamed mnprA gene. The two genes were cloned into plant expressed vector pl301 to transform the transgneic CrylAc tobacco. While no mutant plant was obtained. Maybe because thenprA protein was not specific proteinase.