| Binary-bacterial artificial chromosome (BIBAC) vector, containing both features of BAC vector and Agrobacterium Ti plasmid, not only can function as BAC vector, but also transfer large DNA fragment into plant genome directly. It is also an important tool for gene map-based cloning and functional analysis. Stable maintenance of BIBAC clones in Agrobacterium EHA105 is a prerequisite for rice transformation by BIBAC vector, and BIBAC clones successfully integrated into rice genome provide direct evidence for the feasibility of large DNA fragments genetic transformation within and among species.In the experiment, BIBAC clones with different insert sizes were chosen from our lab’s Zea Mays B73 BIBAC library (4,<50Kb; 4, about 80Kb; 4, about 100Kb; 2, about 130Kb and one, about 160Kb), their stability in Agrobacterium EHA105 were checked, and some of them were transformed into ZhongHua11 using Agrobaterium pools, each containing BIBAC clones of close insert sizes. The results are as follows:1. After the selected BIBAC clones were cultured for 96h in E.coli DH10B, plasmids were extracted, digested with the restriction enzyme I-SceⅠand separated by Pulsed Filed Gel Electrophoresis (PFGE). The results showed no changes of insert sizes, demonstrating that Zea Mays B73 BIBAC clones were still stable after about 200 generations in E. coli.2. In the direct and indirect stability test of BIBAC clones in Agrobacterium, BIBAC plasmids were transferred into Agrobacterium EHA105, single colonies were cultured for 48 h and 96 h (2 X 48 h). In the direct stability test, the digested plasmids with the restriction enzyme I-SceⅠ, extracted from Agrobacterium cultures, were directly analyzed by PFGE. The time of 96 h (4 days) is required for Agrobacterium to finish the co-culture with rice callus in rice transformation. The results showed little changes of insert sizes indicating that BIBAC clones in EHA105 were basically stable in the 48 h and 96 h culturing process. In the indirect method to test BIBAC stability in Agrobacterium, plasmids were extracted from Agrobacterium EHA105 and retransformed into E. coli DH10B, and then plasmids extracted from DH10B, digested with the restriction enzyme Not I were analyzed by PFGE. The results also showed little insert size changes, again indicating that BIBAC clones were stable in Agrobacterium EHA105 at least within the time needed for rice transformation.3. T-DNA complex formation is from right border (RB) to left border (LB) of BIBAC vector, hpt and gus are RB and LB marker genes, respectively. If hpt and gus both show positive in PCR tests of resistant plants, we primarily conclude that T-DNA with large DNA fragment has been intactly integrated into receptor genome. Eighteen BIBAC clones in different Agrobacterium culture pools of close insert sizes were used to transform rice ZhongHua11. In the experiment,33 hpt positive plants were gotten from 111 resistant plants, and 4 gus positive plants were gotten from hpt positive plants. Four hpt and gus positive plants indicate BIBAC could be transferred to rice genome completely.4. End-sequences of some insert DNA fragments of Zea Mays (ie. inward flanking sequences of the two vector arms) from rice positive transgenic plants were obtained by TAIL-PCR. The No.5 positive plant was confirmed to be from rice transformation of BIBAC b6 plasmid through comparisons of the Tail-PCR sequences with the end-sequences of the 18 BIBAC clones. The consensus sequences of the Tail-PCR sequences of the No.5 positive plant and end-sequences of b6 plasmid were mapped to a region on chromosome 7 of the B73 genome sequence spanning 164 kb, very close to the BIBAC insert size estimated on PFGE of b6 plasmid. Analysis of the region sequence showed that it contains 88.1% of repetitive sequences. Therefore, we conclude that a 164kb, maize B73 genomic DNA fragment with 88.1% repeat sequences, was completely integrated into rice genome using BIBAC vector in Agrobacterium-mediated genetic transformation. |
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