期刊
NEW PHYTOLOGIST
卷 229, 期 5, 页码 2859-2872出版社
WILEY
DOI: 10.1111/nph.17032
关键词
Agrobacterium‐ mediated plant transformation; Arabidopsis thaliana; DNA polymerase θ Oryza sativa (rice); T‐ DNA integration; T‐ DNA junction sequences
资金
- US National Science Foundation
- Purdue Center for Cancer Research via an NIH NCI [P30 CA023168]
- JST, PRESTO Japan [JPMJPR16QA]
- Research Grant from NARO Gender Equality Program
- Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (JSPS)
- Cross-ministerial Strategic Innovation Promotion Program (SIP)
- Program for Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry
The mechanism of T-DNA integration into the plant genome remains controversial, with some evidence suggesting that DNA polymerase theta (PolQ) may play a crucial role. Experimental results with Arabidopsis and rice showed that while polQ mutant plants had a lower transformation frequency compared to wild-type plants, they still generated stable transformants. This implies that T-DNA integration may involve multiple redundant pathways or some as yet unidentified pathway.
Integration of Agrobacterium tumefaciens transferred DNA (T-DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T-DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end-joining (NHEJ) pathways. Recent evidence suggests that in Arabidopsis, DNA polymerase theta (PolQ) may be a crucial enzyme involved in T-DNA integration. We conducted quantitative transformation assays of wild-type and polQ mutant Arabidopsis and rice, analyzed T-DNA/plant DNA junction sequences, and (for Arabidopsis) measured the amount of integrated T-DNA in mutant and wild-type tissue. Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency of polQ mutants was c. 20% that of wild-type plants. T-DNA/plant DNA junctions from these transformed rice and Arabidopsis polQ mutants closely resembled those from wild-type plants, indicating that loss of PolQ activity does not alter the characteristics of T-DNA integration events. polQ mutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation. We suggest that either multiple redundant pathways function in T-DNA integration, and/or that integration requires some yet unknown pathway.
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