GM벼 OsCK1의 도입유전자 구조분석: Whole Genome Shotgun Sequencing을 이용한 다중 도입된 T-DNA 구조 분석
Transgene Structure Analysis of Genetically Modified OsCK1 Rice : Whole Genome Shotgun Sequencing as a Tool for Structure Analysis of Multi-Inserted T-DNAs
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The purpose of this study was to characterize the T-DNAs introduced into the transgenic OsCK rice, as part of abiosafety evaluation. Choline Kinase (CK) gene is upregulated in the transgenic OsCK rice. We identified the insertion sites,flanking sequences, structures and sequences of the inserted T-DNAs. Based on the adaptor-ligation PCR, we found that the rightborder of the T-DNA was inserted at position no. 129971, on BAC clone OSJNBa0014J14 of chromosome 10. The flankingsequences of the left border region of the T-DNA (which was later identified as a region harboring a 1-kb long deleted sequence),could not be identified by various PCR-based trials. However, it was finally identified with whole-genome shotgun sequencing,using an Illumina sequencer. The result indicated that one of the T-DNAs was inserted into the CaMV 35S promoter region,whereas the other T-DNA was introduced at position 128947 on OSJNBa0014J14 clone, with an inverse orientation. During theinsertion process, a 1024-bp-long chromosome sequences flanked by the right border of the T-DNA region was deleted. A 370-bplong left border region and 199-bp long right border region corresponding to the matrix attachment region (MAR) sequences ofthe T-DNA were also deleted. Collectively, these results indicate that whole-genome shotgun sequencing is a useful tool to revealthe detailed sequences and structures of the introduced T-DNAs, especially in the case of multiple T-DNA insertions. Theexpenses incurred on genome sequencing can be easily compensated by minimizing the time and efforts invested in conventionalmolecular analyses.
Abstract
The purpose of this study was to characterize the T-DNAs introduced into the transgenic OsCK rice, as part of abiosafety evaluation. Choline Kinase (CK) gene is upregulated in the transgenic OsCK rice. We identified the insertion sites,flanking sequences, structures and sequences of the inserted T-DNAs. Based on the adaptor-ligation PCR, we found that the rightborder of the T-DNA was inserted at position no. 129971, on BAC clone OSJNBa0014J14 of chromosome 10. The flankingsequences of the left border region of the T-DNA (which was later identified as a region harboring a 1-kb long deleted sequence),could not be identified by various PCR-based trials. However, it was finally identified with whole-genome shotgun sequencing,using an Illumina sequencer. The result indicated that one of the T-DNAs was inserted into the CaMV 35S promoter region,whereas the other T-DNA was introduced at position 128947 on OSJNBa0014J14 clone, with an inverse orientation. During theinsertion process, a 1024-bp-long chromosome sequences flanked by the right border of the T-DNA region was deleted. A 370-bplong left border region and 199-bp long right border region corresponding to the matrix attachment region (MAR) sequences ofthe T-DNA were also deleted. Collectively, these results indicate that whole-genome shotgun sequencing is a useful tool to revealthe detailed sequences and structures of the introduced T-DNAs, especially in the case of multiple T-DNA insertions. Theexpenses incurred on genome sequencing can be easily compensated by minimizing the time and efforts invested in conventionalmolecular analyses.
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