Advertisement

Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Determination of genomic location and structure of the transgenes in marker-free rice-based cholera vaccine by using whole genome resequencing approach

  • 424 Accesses

  • 7 Citations

Abstract

We previously developed a molecularly uniform rice-based oral cholera vaccine (MucoRice-CTB) by using an overexpression system for modified cholera toxin B-subunit, CTB (N4Q) with RNAi to suppress production of the major rice endogenous storage proteins. To establish MucoRice-CTB for human use, here we developed hygromycin phosphotransferase selection marker-free MucoRice-CTB by using two different Agrobacterium tumefaciens, each carrying a distinct T-DNA for co-transformation. In the marker-free candidates from co-transformants by segregation in the seed progeny, we selected a line with high CTB expression, line 51A, which we advanced to the T6 generation by self-pollination to obtain a homozygous line without down-regulation of CTB expression. Southern blot analyses showed that three copies of the CTB gene, but not the backbone of the T-DNA binary vector, were inserted into the rice genome of MucoRice-CTB line 51A. By whole genome resequencing, we showed that the transgenes in this line were inserted into intergenic regions in chromosome 3 and chromosome 12. We determined that two full-length copies, each containing the CTB and RNAi expression cassettes, were inserted in a tandem reverted orientation into chromosome 3. An additional copy of the CTB over-expression cassette with a truncated RNAi cassette was inserted into chromosome 12. These findings provide useful information for the establishment of a seed bank of marker-free MucoRice-CTB for human use.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

Ab:

Antibody

CT:

Cholera toxin

CTB:

Cholera toxin B-subunit

ELISA:

Enzyme-linked immunosorbent assay

MS:

Mass spectrometry

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

RNAi:

RNA interference

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

T-DNA:

Transfer DNA

TAIL-PCR:

Thermal asymmetric interlaced polymerase chain reaction

WT:

Wild type

References

  1. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657

  2. Chen S, Jin W, Wang M, Zhang F, Zhou J, Jia Q, Wu Y, Liu F, Wu P (2003) Distribution and characterization of over 1000 T-DNA tags in rice genome. Plant J 36:105–113

  3. Dale PJ, Clarke B, Fontes EM (2002) Potential for the environmental impact of transgenic crops. Nat Biotechnol 20:567–574

  4. De Block M, Debrouwer D (1991) Two T-DNA’s co-transformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theor Appl Genet 82:257–263

  5. De Buck S, Jacobs A, Van Montagu M, Depicker A (1999) The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J 20:295–304

  6. Ebinuma H, Sugita K, Matsunaga E, Yamakado M (1997) Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci USA 94:2117–2121

  7. EMA (2008) Guideline on the quality of biological active substances produced by stable transgene expression in higher plants. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003154.pdf

  8. Endo S, Sugita K, Sakai M, Tanaka H, Ebinuma H (2002) Single-step transformation for generating marker-free transgenic rice using the ipt-type MAT vector system. Plant J 30:115–122

  9. FDA (2002) Guidance for industry: drugs, biologics, and medical devices derived from bioengineered plants for use in humans and animals. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm124811.pdf

  10. Gelvin SB (2010) Plant proteins involved in Agrobacterium-mediated genetic transformation. Annu Rev Phytopathol 48:45–68

  11. Gheysen G, Herman L, Breyne P, Gielen J, Van Montagu M, Depicker A (1990) Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum. Gene 94:155–163

  12. Gheysen G, Villarroel R, Van Montagu M (1991) Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev 5:287–297

  13. Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994

  14. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301

  15. Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174

  16. Krizkova L, Hrouda M (1998) Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. Plant J 16:673–680

  17. Kuroda M, Kimizu M, Mikami C (2010) A simple set of plasmids for the production of transgenic plants. Biosci Biotechnol Biochem 74:2348–2351

  18. Kurokawa S, Nakamura R, Mejima M, Kozuka-Hata H, Kuroda M, Takeyama N, Oyama M, Satoh S, Kiyono H, Masumura T et al (2013) MucoRice-cholera toxin B-subunit, a rice-based oral cholera vaccine, down-regulates the expression of alpha-amylase/trypsin inhibitor-like protein family as major rice allergens. J Proteome Res 12:3372–3382

  19. Li G, Zhou Z, Liu G, Zheng F, He C (2007) Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae. Curr Genet 51:233–243

  20. Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681

  21. Matzke MA, Mette MF, Matzke AJ (2000) Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol 43:401–415

  22. Mayerhofer R, Koncz-Kalman Z, Nawrath C, Bakkeren G, Crameri A, Angelis K, Redei GP, Schell J, Hohn B, Koncz C (1991) T-DNA integration: a mode of illegitimate recombination in plants. EMBO J 10:697–704

  23. Nandy S, Srivastava V (2012) Marker-free site-specific gene integration in rice based on the use of two recombination systems. Plant Biotechnol J 10:904–912

  24. Nanto K, Sato K, Katayama Y, Ebinuma H (2009) Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants. Plant Cell Rep 28:777–785

  25. Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, Nakanishi U, Matsumura A, Uozumi A, Hiroi T et al (2007) Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci USA 104:10986–10991

  26. Nochi T, Yuki Y, Katakai Y, Shibata H, Tokuhara D, Mejima M, Kurokawa S, Takahashi Y, Nakanishi U, Ono F et al (2009) A rice-based oral cholera vaccine induces macaque-specific systemic neutralizing antibodies but does not influence pre-existing intestinal immunity. J Immunol 183:6538–6544

  27. Oyama M, Kozuka-Hata H, Tasaki S, Semba K, Hattori S, Sugano S, Inoue J, Yamamoto T (2009) Temporal perturbation of tyrosine phosphoproteome dynamics reveals the system-wide regulatory networks. Mol Cell Proteomics 8:226–231

  28. Parkhi V, Rai M, Tan J, Oliva N, Rehana S, Bandyopadhyay A, Torrizo L, Ghole V, Datta K, Datta SK (2005) Molecular characterization of marker-free transgenic lines of indica rice that accumulate carotenoids in seed endosperm. Mol Genet Genomics 274:325–336

  29. Ramana Rao MV, Parameswari C, Sripriya R, Veluthambi K (2011) Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene. Plant Cell Rep 30:1241–1252

  30. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

  31. Sha Y, Li S, Pei Z, Luo L, Tian Y, He C (2004) Generation and flanking sequence analysis of a rice T-DNA tagged population. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 108:306–314

  32. Srivastava V, Ariza-Nieto M, Wilson AJ (2004) Cre-mediated site-specific gene integration for consistent transgene expression in rice. Plant Biotechnol J 2:169–179

  33. Subbaiyan GK, Waters DL, Katiyar SK, Sadananda AR, Vaddadi S, Henry RJ (2012) Genome-wide DNA polymorphisms in elite indica rice inbreds discovered by whole-genome sequencing. Plant Biotechnol J 10:623–634

  34. Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H (2006) Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J 47:969–976

  35. Tokuhara D, Yuki Y, Nochi T, Kodama T, Mejima M, Kurokawa S, Takahashi Y, Nanno M, Nakanishi U, Takaiwa F et al (2010) Secretory IgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine. Proc Natl Acad Sci USA 107:8794–8799

  36. Tzfira T, Li J, Lacroix B, Citovsky V (2004) Agrobacterium T-DNA integration: molecules and models. Trends Genet 20:375–383

  37. Xu X, Liu X, Ge S, Jensen JD, Hu F, Li X, Dong Y, Gutenkunst RN, Fang L, Huang L et al (2012) Resequencing 50 accessions of cultivated and wild rice yields markers for identifying agronomically important genes. Nat Biotechnol 30:105–111

  38. Yamakawa H, Hirose T, Kuroda M, Yamaguchi T (2007) Comprehensive expression profiling of rice grain filling-related genes under high temperature using DNA microarray. Plant Physiol 144:258–277

  39. Yuki Y, Mejima M, Kurokawa S, Hiroiwa T, Takahashi Y, Tokuhara D, Nochi T, Katakai Y, Kuroda M, Takeyama N et al (2013) Induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin B subunit vaccine without plant-associated sugar modification. Plant Biotechnol J 11:799–808

Download references

Acknowledgments

We are grateful to Drs. Masaaki Oyama, Hiroko Kozuka-Hata, Satoshi Kaneto, Shintaro Sato, and Mr. Yuji Suzuki for useful discussions and technical support. This work was supported by Grants from the Programs of Special Coordination Funds for Promoting Science and Technology and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y. Y., H. K.); the Ministry of Health, Labor and Welfare of Japan (Y. Y., H. K.); the New Energy and Industrial Technology Development Organization (NEDO) (H. K.); and the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-step); and the Research and Development Program for New Bio-industry Initiatives of the Bio-oriented Technology Research Advancement Institution (Y. Y.).

Conflict of interest

None.

Author information

Correspondence to Yoshikazu Yuki.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 344 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mejima, M., Kashima, K., Kuroda, M. et al. Determination of genomic location and structure of the transgenes in marker-free rice-based cholera vaccine by using whole genome resequencing approach. Plant Cell Tiss Organ Cult 120, 35–48 (2015). https://doi.org/10.1007/s11240-014-0575-4

Download citation

Keywords

  • Selection marker-free
  • Gene location
  • MucoRice-CTB
  • Oral vaccine
  • Oryza sativa
  • Cholera