An agrobacterium tumefaciens-mediated co-tr- ansformation method was established and successfully used to transfer an antisense waxy gene into rice. The binary vec- tor p13W4 contains an antisense waxy gene, a hygromy...An agrobacterium tumefaciens-mediated co-tr- ansformation method was established and successfully used to transfer an antisense waxy gene into rice. The binary vec- tor p13W4 contains an antisense waxy gene, a hygromycin resistance gene (hpt) and a report gene (gus). The other bi- nary vector p13W8 contains only an antisense waxy gene in the T-DNA region. Two separate strains of A. tumefaciens, containing p13W4 and p13W8 mixed at a ratio of 1︰9, were used to transform a high-yield rice strain Chao 2-10. A total of 34 transgenic plants were obtained. PCR analysis showed that 15 of them were co-transformed. After GUS staining, the T1seeds with GUS positive were eliminated. The GUS nega- tive T1 seeds were germinated, and the seedlings were ana- lyzed for the existence of antisense waxy gene. The transgenic T1 plants with only antisense waxy gene further were con- firmed through Southern blot analysis. The T2 seeds collected from marker-free T1 plants were used for amylose content analysis. The results showed that the amylose content in marker-free transgenic seeds reduced by up to 28.61% over the wild type rice. This marker-free transgenic rice with low amylose contents obtained in this project could be used to screen soft grain, and could also be used as a new rice re- source for eating quality improvement.展开更多
基金the Shanghai Science and Technology Development Fund(Grant No.023112027).
文摘An agrobacterium tumefaciens-mediated co-tr- ansformation method was established and successfully used to transfer an antisense waxy gene into rice. The binary vec- tor p13W4 contains an antisense waxy gene, a hygromycin resistance gene (hpt) and a report gene (gus). The other bi- nary vector p13W8 contains only an antisense waxy gene in the T-DNA region. Two separate strains of A. tumefaciens, containing p13W4 and p13W8 mixed at a ratio of 1︰9, were used to transform a high-yield rice strain Chao 2-10. A total of 34 transgenic plants were obtained. PCR analysis showed that 15 of them were co-transformed. After GUS staining, the T1seeds with GUS positive were eliminated. The GUS nega- tive T1 seeds were germinated, and the seedlings were ana- lyzed for the existence of antisense waxy gene. The transgenic T1 plants with only antisense waxy gene further were con- firmed through Southern blot analysis. The T2 seeds collected from marker-free T1 plants were used for amylose content analysis. The results showed that the amylose content in marker-free transgenic seeds reduced by up to 28.61% over the wild type rice. This marker-free transgenic rice with low amylose contents obtained in this project could be used to screen soft grain, and could also be used as a new rice re- source for eating quality improvement.
