Agrobacterium Mediated Construction Of Rice Mutant Population With Transposon

Transposon tagging is a powerful tool for the study of gene function,especially for isolating genes among different plants including Arabidopsis,tabacco and tomato etc. This effective strategy for gene isolation has beenapplied to rice and progress has been made in recent years.We developed a rapid and high-efficient rice transformation techniquemediated by Agrobacterium tumefaciens and used it to construct a mutant pooi.Immature and mature embryos of rice (Oryza sativa L. subsp. Japonica cv.Zhonghua No. 11) were used to initiate embryogenic calli. Calli were thenco-cultured with A. tumefaciens strain EHA1O5 that harbored Ti plasmidpDsBarl300H or plasmid pUBJTs. The pDsBarl300H carried maize Ds(Dissociation) and Bar gene. And pUBJTs carried Ac transposase (AcTpase)and hygromycin resistance gene. Many independent transgenic lines wereobtained, among which three hundred and eighty seven independenttransgenic lines were identified based on morphological characters andmolecular analysis in this study. In order to induce transposition of theinserted Ds elements, the Ds-inserted transgenic plants with a single siteinsertion were crossed with the transgenic plants carrying AcTPase. Apopulation of about 354 hybrids consisting of AcxDs and/or DsxAc wasconstructed in this study. The Ds element transposition activity was evaluatedand the DNA flanking sequences of Ds insertion sites and reinsertion siteswere analyzed.1.PCR and Southern blotting analysis revealed that the maize transposableelement Ac/Ds had been integrated into the rice genome, and the Ac/Dswas present in the T1 generation. Southern blotting analysis for 96independent transgenic plants indicated that about 63.5% had only oneinsertion site and 20.8% had two.2.Herbicide Basta was applied to To transgenic rice and T1 population withinserted Ds and Bar genes to identify Basta-resistant plants. Threehundred sixty four To independent transgenic plants were sprayed with790.027% Basta, and 95% plants tested were Basta-resistant. Basta wasalso sprayed over the T1 population seedling, and 185 ofT1 lines (66.3%)segregated at 3R: iS ratio, 39 lines (14.0%) at 15R: 15 ratio. Based onthe Basta resistance segregation ratio, the number of Ds element insertionsite was estimated. It showed the similar result as that by Southernblotting analysis. Our data suggested that transgenic rice regeneratedfrom the same callus did not always derived from a same transgenicevent.3.Inverse polymerase chain reaction (Inverse PCR) was used to isolate theDNA sequences flanking the Ds (T-DNA) element. Some transgenicplants had the same integrate site. For examples, both T276-1 and T287possessed the same DNA flanking the Ds element as T434-2. Theflanking sequence of Ds in 1331-2 was as same as in T220a- 1. About65.3% had the different integrate sites. Some evidences indicated that Dselement could be integrated into the protein coding regions. For anexample, in transgenic rice 1369, its insertion site located inglucose-6-phosphate translocator gene.4.The excision frequency of Ds element in cross-derived population wasevaluated with PCR amplification. In the F2 population of DsxAc, 132out of 395 single hybrid plants consisting of both Ds and Ac wereidentified. Thirty individual progenies of them were demonstrated thatDs was excised and transposed to new sites on chromosomes. The resultindicated that the excision frequency of Ds element trans-activated by Actransposase was about 22.7%. Nine DNA fragments tagged with Dselement were isolated and cloned. Two DNA fragments flanking the Dselement sequences did not show similarity to any sequences in GenBank,the accession numbers of them are AF355 153 and AF355770.5.In transgenic To lines and T~ generations, some plants weremorphologically ab...