The Verification of DNA Sequences Adjacent to LB of T-DNA in Transgenic Dendrobium ‘Sonia’ Earsakul Genome Using AL-PCR Technique

Authors

  • Pattama Sonsaka Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140
  • Piyanuch Sornchai สำนักวิจัยพัฒนาเทคโนโลยีชีวภาพ กรมวิชาการเกษตร
  • Parichart Burns
  • Sontichai Chanprame
  • Sermsiri Chanprame

DOI:

https://doi.org/10.14456/thaidoa-agres.2021.11

Keywords:

Transgenic plant, AL-PCR, insertion position

Abstract

In Agrobacterium-mediated genetic transformation, the integration site of T-DNA in plant genome was non-predictability. The inserted position of transgenes may affect gene expression and subsequent physiological traits. This research explained the utilization of Adaptor Ligation-Polymerase Chain Reaction (AL-PCR) technique to verify the sequence adjacent tothe leftborderofT-DNA (LB T-DNA)of the transgenic Dendrobium ‘Sonia’ Earsakul possesses antisense CP-ACO1. The genomic DNA of transgenic orchid was digested with restriction enzyme PsiI to generate blunt ended DNA strands prior to ligating with the blunt end adapter. The PCR was performed using adapter ligated DNA fragments as the templates. The forward primer was designed to be complimentary with adapter sequences and reverse primer was specific to known sequence of hpt gene that was inserted into the plant genome. The analysis of amplicons resulted from AL-PCR technique revealed 3 DNA bands at the sizes of approximately 750, 1,500 and 2,000 base pairs. The DNA sequence analysis of 750 bp band revealed that part of hpt gene in T-DNA was connected to LB sequence that fused to the 160 bp upstream of vector backbone sequences. For the bands of 1,500 and 2,000 base pairs, the DNA sequence analysis could not be achieved due to their low purity and amount

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References

ปิยนุช ศรชัย. 2558. การตรวจสอบความปลอดภัยทางชีวภาพของกล้วยไม้สกุลหวายที่ได้รับการถ่ายยีน antisense CP-ACO1 ในระดับโรงเรือน. วิทยานิพนธ์ปริญญาเอก, มหาวิทยาลัยเกษตรศาสตร์.

Abdal-Aziz, S.A., F. Pliego-Alfaro, M.A. Quesada and J.A. Mercado. 2006. Evidence of frequent integration of non-T-DNA vector backbone sequences in transgenic strawberry plants. J. Biosci. Bioeng. 101(6); 508-510.

Afolabi, A.S., B. Worland, J.W. Snape and P. Vain. 2004. A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations. Theoretical and Applied Genetics. 109: 815–826.

Choi, Y.I, E.W. Noh, M.S. Han and S.H. Son. 1999. Adaptor-aided PCR to identify T-DNA junctions in transgenic plants. BioTechniques. 27: 222-226.

Cullen, D., W. Harwood, M. Smedley, H. Davies and M. Taylor. 2011. Comparison of DNA Walking Methods for Isolation of Transgene-Flanking Regions in GM Potato. Mol Biotechnol. 49(1):19-31.

De Buck, S.D., C.D. Wilde, M.V. Montagu and A. Depicker. 2000. T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Molecular Breeding. 6: 459-468.

Fu, D., P.C.St. Amand, Y. Xiao, S. Muthukrishnan and G.H. Liang. 2006. Characterization of T-DNA integration in creeping bentgrass. Plant Science. 170: 225–237.

Kohli, A., B. Miro and R.M. Twyman. 2010. Transgene integration, expression and stability in plants: strategies for improvements. Transgenic Crop Plants. 201-237.

Kononov, M.E., B. Bassuner and S.B. Gelvin. 1997. Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. The Plant J. 11(5): 945-957.

Kuraya, Y., S. Ohta and M. Fukude. 2004. Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeat in vector for transformation of higher plants mediated by Agrobacterium tumefaciens. Mol Breed. 14:309-320.

Malley, R.C.O., J.S. Alonso, C.J. Kim, T.J. Leisse and J.R. Ecker. 2007. An adapter ligation-mediated PCR method for high throughput mapping of T-DNA inserts in the Arabidopsis genome. NATURE PROTOCOL. 2(11): 2910-2917.

Meza, T.J., B. Stangeland, I.S. Mercy, M. Skarn, D.A. Nymoen, A. Berg, M.A. Butenko, A.M. Hakelian, C. Haslekas, L.A. Meza-Zepeda and R.B. Aalen. 2002. Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences, short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing. Nucleic Acids Res. 30(20): 4556-4566.

Nicolia, A., N. Ferradini, F. Veronesi and D. Rosellini. 2017. An insight into T-DNA integration

events in Medicago sativa. Int. J. Mol. Sci. 18: 1951-1966.

Okwuonu, I.C., C.N. Egesi and N.J. Taylor. 2019. Vector backbone integration in transgenic cassava is significantly correlated to T-DNA copy number. Nig. J. Biotech. 36(2): 77-86.

Oltmanns, H., B. Frame, L.Y. Lee, S. Johnson, B. Li, K. Wang and S.B. Gelvin. 2010. Generation of Backbone-free, Low transgene copy plants by launching T-DNA from the Agrobacterium chromosome. Plant Physiol. 152: 1158-1166.

Padegimas, L.S. and N.A. Reichert. 1998. Adaptor ligation-based polymerase chain reaction mediated walking. Anal Biochem. 260: 149-153.

Petti, C., T. Wendt, C. Meade and E. Mullins. 2009. Evidence of genotype dependency within Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in transgenic Phytophthora infestans-tolerant potato. J. Biosci. Bioeng. 107(3): 301-306.

Sha, Y., S. Li, Z. Pei, L. Luo, Y. Tian and C. He. 2004. Generation and flanking sequence analysis of rice T-DNA tagged population. Theoretical and Applied Genetics, 108: 306-314.

Sornchai, P., R. Koto, P. Burns, S. Chanprame, W. Imsabai and S. Chanprame. 2015. Genetic transformation of Dendrobium ‘Sonia Earsakul’ with antisense Carica papaya ACO1 gene. Mod. Appl. Sci. 9(12): 125-133.

Sornchai, P., W.G. vanDoorn, W. Imsabai, P. Burns and S. Chanprame. 2020. Dendrobium orchids carrying antisense ACC oxidase: Small changes in flower morphology and a delay of bud abortion, flower senescence, and abscission of flowers. Transgenic Res. 29.429-442.

Spertini, D., C. Beliveau and G. Bellemare. 1999. Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA. Biotechniques. 27: 308-314.

Yang, X., F. Li, X. Zhang, K. Liu, Q. Wang, C. Zhang, C. Liu, W. Zhu, G. Shan, C.K. Chin and W. Fang. 2013. Integration and characterization of T-DNA insertion in Upland Cotton. Czech j. Genet. Plant Breed. 49(2): 51-57.

Zeng, F., Y. Zhan, H. Zhao, Y. Xin, H. Qi and C. Yang. 2010. Molecular characterization of T-DNA integration sites in transgenic birch. Trees. 24: 753–762.

Zhang, J., C. Lin, J.Q. Cheng, H.Z. Mao, X.P. Fan, Z.H. Meng, K.M. Chan, H.J. Zhang, J.F. Qi, L.H. Ji and Y. Hong. 2008. Transgene integration and organization in cotton (Gossypium hirsutum L.) genome. Transgenic Res. 17: 293-306.

Zheng, S.J., B. Henken, Sofiari, E. Jacobsen, F.A. Krens and C. Kik. 2001. Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders. Transgenic Res. 10: 237–245.

Ziemienowicz, A., T. Tzvi and B. Hohn. 2008. Mechanisms of T-DNA integration. p.395-440. In: Agrobacterium: From Biology to Biotechnology; Tzvi Tzfira and Vitaly Citrovsky, Ed.; Springer New York p.395-440.

Published

2021-05-04

How to Cite

Sonsaka ป. ., Sornchai, P., Burns ป., Chanprame ส. ., & Chanprame เ. (2021). The Verification of DNA Sequences Adjacent to LB of T-DNA in Transgenic Dendrobium ‘Sonia’ Earsakul Genome Using AL-PCR Technique. Thai Agricultural Research Journal, 39(2). https://doi.org/10.14456/thaidoa-agres.2021.11

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Section

Technical or research paper