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. Documents Reimbursable with CSREES. Log 33063.
This report documents research conducted under funding from the USDA Biotechnology Risk Assessment Grants program. The goal of the project is to develop the molecular tools to genetically engineer rice using only native rice DNA sequences (e.g. to create “intragenic” rice). In this first year of the project, a post-doctoral research scientist was successfully recruited and hired mid-year. Various components necessary for implementation of the project including the rice-derived gene transfer sequences (required for Agrobacterium transformation), several promoters with constitutive or pollen-specific expression patterns in rice, the intragenic and GUS reporter genes, the selectable marker gene and the Cre site-specific recombinase gene sequence, have been isolated and sequence confirmed. These components have been assembled into four intragenic transformation binary vector constructs. Molecular analysis has confirmed that each of the novel vectors contain the assorted components in their desired orientation. Transient expression assays have validated that the rice-derived reporter gene cassette elicits anthocyanin accumulation in rice leaf cells, demonstrating that the promoter-reporter gene fusion is functional. Preliminary experiments have been performed to examine the ability of Agrobacterium to transform rice with the new intragenic transformation vectors. The results indicate that Agrobacterium successfully mediates translocation and detectable transient expression of the intragenic constructs in rice cells. Research further examining the DNA transfer efficiency from these vectors and their ability to generate stably transformed intragenic rice plants has been initiated. This research contributes to Component 3 of National Program 302 – Plant Biotechnology Risk Assessment, chiefly to Problem Statement 3A, Improving and Assessing Genetic Engineering Technology.
Grant project progress is monitored on a daily basis via informal communication with the staff conducting the research. Formal review of research results is performed periodically during laboratory group meetings.