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| The hygromycin-resistant gene (HPH) was successfully co-transformed with the self-complementary chimeric RNA/DNA oligonucleotide to develop a site-specific deletion mutantby means of particle bombardment. Two COs, CO1 for deletion and CO2 for insertion at the coding sequence of cytochrome P450 gene CYP81A6, respectively, were designed to target the nucleotide of the endogenous gene in rice. Using the CO1 and HPH, 94 calli regenerated plants from 207 hygromycin resistant calli were obtained, but all of the transgenic plants were sensitive to the bentazon, the results show that no conversion was mediated by the CO1. As to the CO2, 567 independent plants were regenerated from 823 independent hygromycin-resistant calli. Herbicide test of the T1 transgenic plants indicated that 2 plants from 2 different lines were sensitive to the herbicide. PCR products of the sensitive plants digested by Nae1 further demonstrated that the two independent events were modified in the target site, and sequencing of the PCR products was also confirmed it. The frequency of gene conversion in the resistant rice was estimated to be approximately 3.5×10-3. The results demonstrated that any genes in rice can be precisely modified at the nucleotide level by co-transformation of COs and the hygromycin-resistant gene. |
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| Keywords:Rice;self-complementary chimeric RNA/DNA oligonucleotide;HPH;co-transformationkey |
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