1a.Objectives (from AD-416)
The goal of the project is to develop and characterize the molecular tools required to generate genetically engineered intragenic rice plants. Two Agrobacterium transformation binary vectors carrying Plant-derived transfer DNAs (P-DNAs) will be constructed. A compatible helper binary vector carrying a separate Transfer-DNA (T-DNA) with a selectable marker, a reporter gene, and a cre recombinase expression cassette, all flanked by loxP attachment sites will also be constructed. The ability of this dual binary vector Agrobacterium mediated transformation system to generate intragenic rice plants will be evaluated.
1b.Approach (from AD-416)
The molecular tools needed to generate genetically engineered intragenic rice will be identified and tested. A dual binary vector system with separate P-DNAs and T-DNAs will be constructed using standard molecular biology techniques. The intragenic vector will carry a P-DNA that contains only native rice-derived sequences that function as Agrobacterium border sequences, expression control elements (promoters and terminators) and one of two rice regulatory genes which stimulate the production of colored anthocyanins or proanthocyanidins in various rice tissues. A second binary vector compatible with the intragenic vector will also be constructed. This vector will contain standard Agrobacterium T-DNA border sequences and three transgene expression cassettes; a hygromycin resistance selectable marker, a constitutively expressed GUS reporter, and the cre recombinase gene controlled by a rice anther-specific promoter. These transgenes present on the T-DNA will be flanked by loxP attachment sites allowing Cre-mediated site-specific excision and transgene removal in rice anther and pollen tissue. This transformation system also incorporates an alternative strategy to identify marker-free intragenic rice plants due to the presence of separate T-DNA and P-DNA transfer from Agrobacterium. Some of the genetically engineered rice will contain a P-DNA that has integrated into the rice genome at locus separate from the integrated T-DNA. In these circumstances, some of the progeny plants will contain only the P-DNA due to independent segregation. The efficiency by which these two methods produce marker-gene free rice plants positive for anthocyanin accumulation will be evaluated and compared.
The goal of this agreement is to develop efficient strategies that mitigate potential risks associated with the biotechnological improvement of rice, which contributes to objective 1 of the in-house project. Specifically, novel molecular tools to genetically engineer rice using only native rice DNA sequences (i.e. to create “intragenic” rice) were constructed and tested. A transformation vector containing a native rice selectable marker gene and a rice gene which activates the production of a visible red color in certain parts of the plant was assembled and utilized for genetic engineering. Numerous transformed rice plants were produced that expressed both the selected and unselected rice genes. However, molecular examination detected the unwanted presence of non-rice DNA sequences that were derived from the transformation vector. To mitigate this undesirable outcome, alternative strategies and new transformation constructs were designed. Experiments examining the functionality of these alternative approaches and their ability to increase the frequency of the production of intragenic rice plants that contain only native DNA sequences are underway.