Transformation of rice with large maize genomic DNA fragments containing high content repetitive sequences
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Large and complex maize BIBAC inserts, even with a length of about 164 kb and repeat sequences of 88.1 %, were transferred into rice.
The BIBAC vector has been established to clone large DNA fragments and directly transfer them into plants. Previously, we have constructed a maize B73 BIBAC library and demonstrated that the BIBAC clones were stable in Agrobacterium. In this study, we demonstrated that the maize BIBAC clones could be used for rice genetic transformation through Agrobacterium-mediated method, although the average transformation efficiency for the BIBAC clones (0.86 %) is much lower than that for generally used binary vectors containing small DNA fragments (15.24 %). The 164-kb B73 genomic DNA insert of the BIBAC clone B2-6 containing five maize gene models and 88.1 % of repetitive sequences was transferred into rice. In 18.75 % (3/16) of the T1, 13.79 % (4/29) of the T2, and 5.26 % (1/19) of the T3 generation transgenic rice plants positive for the GUS and HYG marker genes, all the five maize genes can be detected. To our knowledge, this is the largest and highest content of repeat sequence-containing DNA fragment that was successfully transferred into plants. Gene expression analysis (RT-PCR) showed that the expression of three out of five genes could be detected in the leaves of the transgenic rice plants. Our study showed a potential to massively use maize genome resource for rice breeding by mass transformation of rice with large maize genomic DNA fragment BIBAC clones.
KeywordsBIBAC Large DNA fragment Genetic transformation Agrobacterium Rice Maize
Bacterial artificial chromosome
Basic local alignments search tool
Transformation-competent artificial chromosome
Pulsed-field gel electrophoresis
We thank Dr. Carol M. Hamilton and the Cornell Center for Technology Enterprise and Commercialization for providing the help for vector pCH32 and pCH30. This work was supported by the National Programs for High Technology Research and Development (863 Project: 2012AA10A305) and the National Natural Science Foundation of China (Grant No. 30971748).
Conflict of interest
The authors declare that they have no conflict of interest.
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