Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality of indica hybrid rice is closely related to the high amylose level in rice grains. In order to ...Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality of indica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of the indica hybrid rice by genetic engineering, an antisense fragment of rice waxy gene, driven by the 5’-franking sequences of the rice waxy gene, was successfully introduced into three major parent lines of indica hybrid rice, all contain a high amylose level in the grains, via Agrobacte-rium, and more than 100 hygromycin-resistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisense waxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains展开更多
Insertional mutagenesis based on maize Activator/Dissociator (Ac/Ds) transposons is becoming a ma- jor approach used to produce a saturated mutant collection in rice. In this research, Ds-T-DNA trans- formed homozygot...Insertional mutagenesis based on maize Activator/Dissociator (Ac/Ds) transposons is becoming a ma- jor approach used to produce a saturated mutant collection in rice. In this research, Ds-T-DNA trans- formed homozygotes were crossed with Ac-T-DNA transformed homozygotes in order to establish an Ac/Ds transposon system in rice. The successive investigation of Ds transposition from F1 to F5 gen- erations indicated that the frequencies of germinal transposition increased over successive genera- tions and reached 54.2% in F3 generation. The Ds transposition pattern revealed that a Ds transposition induced an approximately 170-bp deletion of T-DNA sequence and another Ds transposition carried a 272-bp T-DNA sequence. Using thermal asymmetric interlaced PCR (TAIL-PCR), some flanking se- quences of the Ds element were amplified. Analyses of 17 Ds-flanking sequences showed that five Ds were inserted into gene regions. The Ds could transpose not only to the linked sites but also to the unlinked sites. The frequency of inter-chromosomal transposition of Ds was 33.3%.展开更多
Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agro-bacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from ind...Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agro-bacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from independent transformants and classified into six main types on the basis of the origin of filler DNA between the right border of T-DNA and flanking sequence of rice genome. Type I sequences were the most common and showed canonical in-tegration that T-DNA right border was followed by rice genome sequence with or without filler DNA of no more than 50 bp, while type II sequences displayed a vector-genome combination that T-DNA right border was followed by a vector fragment and then connected with rice genome sequence. The location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using BLAST analysis. The 340 Ds insertions at an average in-terval of 0.8 megabase (Mb) constructed a basic framework of Ds starter points on whole rice chromosomes. The frequency of T-DNA (Ds) inserted into the exons of predicted genes on chromosome one was 21%. Knowledge of T-DNA (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis.展开更多
基金This work was supported by the State "863" High-Tech Project (Grant No. 2001AA212101)the Chinese Department of Science and Technology (Grant No. J99-A-005)High-Tech Project of Jiangsu Province (Grant No. BG2001302).
文摘Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality of indica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of the indica hybrid rice by genetic engineering, an antisense fragment of rice waxy gene, driven by the 5’-franking sequences of the rice waxy gene, was successfully introduced into three major parent lines of indica hybrid rice, all contain a high amylose level in the grains, via Agrobacte-rium, and more than 100 hygromycin-resistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisense waxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains
基金Supported by National Basic Research Program of China (Grant No. G19990116)
文摘Insertional mutagenesis based on maize Activator/Dissociator (Ac/Ds) transposons is becoming a ma- jor approach used to produce a saturated mutant collection in rice. In this research, Ds-T-DNA trans- formed homozygotes were crossed with Ac-T-DNA transformed homozygotes in order to establish an Ac/Ds transposon system in rice. The successive investigation of Ds transposition from F1 to F5 gen- erations indicated that the frequencies of germinal transposition increased over successive genera- tions and reached 54.2% in F3 generation. The Ds transposition pattern revealed that a Ds transposition induced an approximately 170-bp deletion of T-DNA sequence and another Ds transposition carried a 272-bp T-DNA sequence. Using thermal asymmetric interlaced PCR (TAIL-PCR), some flanking se- quences of the Ds element were amplified. Analyses of 17 Ds-flanking sequences showed that five Ds were inserted into gene regions. The Ds could transpose not only to the linked sites but also to the unlinked sites. The frequency of inter-chromosomal transposition of Ds was 33.3%.
文摘Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agro-bacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from independent transformants and classified into six main types on the basis of the origin of filler DNA between the right border of T-DNA and flanking sequence of rice genome. Type I sequences were the most common and showed canonical in-tegration that T-DNA right border was followed by rice genome sequence with or without filler DNA of no more than 50 bp, while type II sequences displayed a vector-genome combination that T-DNA right border was followed by a vector fragment and then connected with rice genome sequence. The location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using BLAST analysis. The 340 Ds insertions at an average in-terval of 0.8 megabase (Mb) constructed a basic framework of Ds starter points on whole rice chromosomes. The frequency of T-DNA (Ds) inserted into the exons of predicted genes on chromosome one was 21%. Knowledge of T-DNA (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis.
