Skip to main content
SpringerLink
Log in

Development of insect-resistant transgenic cotton with chimeric TVip3A* accumulating in chloroplasts

  • Letter to the Editor
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

An optimized vip3A gene, designated as vip3A* was chemically synthesized and a thi1 gene chloroplast transit peptide coding sequence was attached to its 5′ end to produce the tvip3A*. vip3A* and tvip3A* genes were transformed into Gossypium hirsutum cv. Zhongmiansuo35. Of 42 independent transformants, 36 were positive for the vip3A* or tvip3A* gene. Four independent transgenic T1 lines with single-copy insertions and unchanged phenotypes (CTV1 and CTV2 for tvip3A*, and CV1 and CV2 for vip3A*) were selected by Southern blotting, and subjected to an insect bioassay and field assessment. Four homozygous T2 transgenic lines were then selected and the amount of expressed Vip3A* protein was determined by western blotting and ELISA. The protein concentrations of CTV1 and CTV2 were about three-fold higher than those of CV1 and CV2. As expected, the Vip3A* protein of CTV1 and CTV2 were transported to the chloroplasts, where they accumulated. The Vip3A* protein concentration in the chloroplasts of CTV1 and CTV2 was about 15-fold of that of CV1 and CV2. All four transgenic lines showed 100% mortality against fall armyworm (Spodoptera frugiperda) and beet armyworm (Spodoptera exigua) by insect bioassay. Moreover, CTV1 and CTV2 exhibited 100% mortality against cotton bollworm (CBW, Helicoverpa zea), whereas CV1 and CV2 showed 75.0% and 72.5% mortality against CBW, respectively. The field bioassay indicated that CTV1 and CTV2 were more resistant to CBW than CV1 and CV2. Our results suggest that the two tvip3A* transgenic lines (CTV1 and CTV2) can be used to develop insect-resistant cultivars and could be used as a resource for raising multi-toxins-expressing transgenic cotton.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Anilkumar KJ, Rodrigo-Simon A, Ferre J, Pusztai-Carey M, Sivasupramaniam S, Moar WJ (2008) Production and characterization of Bacillus thuringiensis Cry1Ac-resistant cotton bollworm Helicoverpa zea (Boddie). Appl Environ Microbiol 74:462–469

    Article  PubMed  CAS  Google Scholar 

  • Bayley C, Trolinder N, Ray C, Morgan M, Quisenberry JE, Ow DW (1992) Engineering 2, 4-D resistance into cotton. Theor Appl Genet 83:5

    Article  Google Scholar 

  • Bommireddy PL, Leonard BR (2008) Survivorship of Helicoverpa zea and Heliothis virescens on cotton plant structures expressing a Bacillus thuringiensis vegetative insecticidal protein. J Econ Entomol 101:1244–1252

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Cao J, Zhao JZ, Tang D, Shelton M, Earle D (2002) Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins. Theor Appl Genet 105:258–264

    Article  PubMed  CAS  Google Scholar 

  • Chabregas SM, Luche DD, Farias LP, Ribeiro AF, van Sluys MA, Menck CF, Silva-Filho MC (2001) Dual targeting properties of the N-terminal signal sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts. Plant Mol Biol 46:639–650

    Article  PubMed  CAS  Google Scholar 

  • Chabregas SM, Luche DD, Van Sluys MA, Menck CF, Silva-Filho MC (2003) Differential usage of two in-frame translational start codons regulates subcellular localization of Arabidopsis thaliana THI1. J Cell Sci 116:285–291

    Article  PubMed  CAS  Google Scholar 

  • Chen JW, Tang LX, Tang MJ, Shi YX, Pang Y (2002) Cloning and expression product of vip3A gene from Bacillus thuringiensis and analysis of inseceicidal activity. Sheng Wu Gong Cheng Xue Bao 18:687–692

    PubMed  CAS  Google Scholar 

  • Cohen MB, Gould F, Bentur JS (2000) Bt rice: practical steps to sustainable use. Int Rice Res Note 25(2):4–10

    Google Scholar 

  • De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74

    Article  PubMed  Google Scholar 

  • Doss VA, Kumar KA, Jayakumar R, Sekar V (2002) Cloning and expression of the vegetative insecticidal protein (vip3 V) gene of Bacillus thuringiensis in Escherichia coli. Protein Expr Purif 26:82–88

    Article  PubMed  CAS  Google Scholar 

  • Environmental Protection Agency (1998) The Environmental Protection Agency’s white paper on Bt plant-pesticide resistance management. Publication 739-S-98–001. US Environmental Protection Agency, Washington, DC

    Google Scholar 

  • Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG (1996) Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci USA 93:5389–5394

    Article  PubMed  CAS  Google Scholar 

  • Ferre J, Van Rie J (2002) Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu Rev Entomol 47:501–533

    Article  PubMed  CAS  Google Scholar 

  • Firoozabady E, DeBoer DL, Merlo DJ, Halk EL, Amerson LN, Rashka KE, Murray EE (1987) Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol Biol 10:105–116

    Article  CAS  Google Scholar 

  • Guo HN, Wu JH, Chen XY, Luo XL, Lu R, Shi YJ, Qin HM, Xiao JL, Tian YC (2003) Cotton plants transformed with the activated chimeric cry1Ac and API-B genes. Acta Botanica Sinica 45(1):108–113

    CAS  Google Scholar 

  • Jackson RE, Marcus MA, Gould F, Bradley JR Jr, Van Duyn JW (2007) Cross-resistance responses of CrylAc-selected Heliothis virescens (Lepidoptera: Noctuidae) to the Bacillus thuringiensis protein vip3A. J Econ Entomol 100:180–186

    Article  PubMed  CAS  Google Scholar 

  • Kurtz RW, McCaffery A, O’Reilly D (2007) Insect resistance management for Syngenta’s VipCot transgenic cotton. J Invertebr Pathol 95:227–230

    Article  PubMed  Google Scholar 

  • Lee MK, Walters FS, Hart H, Palekar N, Chen JS (2003) The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin. Appl Environ Microbiol 69:4648–4657

    Article  PubMed  CAS  Google Scholar 

  • Lee MK, Miles P, Chen JS (2006) Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem Biophys Res Commun 339:1043–1047

    Article  PubMed  CAS  Google Scholar 

  • Li TY, Tian YC, Qing XF (1994) Studies on high-efficient insect resistance transgenic tobacco. China Sinica (B):268-276

  • MacIntosh SC, McPherson SL, Perlak FJ, Marrone PG, Fuchs RL (1990a) Purification and characterization of Bacillus thuringiensis var. tenebrionis insecticidal proteins produced in E. coli. Biochem Biophys Res Commun 170:665–672

    Article  PubMed  CAS  Google Scholar 

  • MacIntosh SC, Stone TB, Sims SR, Hunst PL, Greenplate JT, Marrone PG, Perlak FJ, Fischhoff DA, Fuchs RL (1990b) Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects. J Invertebr Pathol 56:258–266

    Article  PubMed  CAS  Google Scholar 

  • Maqbool SB, Husnain T, Riazuddin S, Masson L, Christou P (1998) Effective control of yellow stem borer and rice leaf folder in transgenic rice indica varieties Basmati 370 and M7 using the novel δ-endotoxin cry2A Bacillus thuringiensis gene. Mol Breed 6:501–507

    Article  Google Scholar 

  • McCaffery A, Capiro M, Jackson R, Marcus M, Martin T, Dickerson D, Negrotto D, O’Reilly D, Chen E, and Lee M (2006) Proceedings of Effective IRM with a novel insecticidal protein, Vip3A. Beltwide cotton conferences, San Antonio, TX National Cotton Council Memphis, TN 2006:1229–1235

  • Mesrati LA, Tounsi S, Jaoua S (2005) Characterization of a novel vip3-type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid. FEMS Microbiol Lett 244:353–358

    Article  PubMed  CAS  Google Scholar 

  • Micinski S, Waltman B (2005) Efficacy of VipCOT for control of the bollworm/tobacco budworm complex in Northwest Louisiana. Proceedings of 2005 Beltwide cotton conferences New Orleans, LA National Cotton Council Memphis, TN January 2005:1239–1242

  • Paterson AH, Brubaker CL, Wendel JF (1993) A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Rep 11:122–127

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Selvapandiyan A, Arora N, Rajagopal R, Jalali SK, Venkatesan T, Singh SP, Bhatnagar RK (2001) Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis. Appl Environ Microbiol 67:5855–5858

    Article  PubMed  CAS  Google Scholar 

  • Sena JA, Hernandez-Rodriguez CS, Ferre J (2009) Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites. Appl Environ Microbiol 75:2236–2237

    Article  PubMed  CAS  Google Scholar 

  • Shelton AM, Zhao JZ, Roush RT (2002) Economic, ecological, food safety, and social consequences of the deployment of bt transgenic plants. Annu Rev Entomol 47:845–881

    Article  PubMed  CAS  Google Scholar 

  • Singh CK, Ojha A, Bhatanagar RK, Kachru DN (2008) Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs–standard single, multiplex and construct-specific PCR assays. Anal Bioanal Chem 390:377–387

    Article  PubMed  CAS  Google Scholar 

  • Tabashnik BE, Carriere Y, Dennehy TJ, Morin S, Sisterson MS, Roush RT, Shelton AM, Zhao JZ (2003) Insect resistance to transgenic Bt crops: lessons from the laboratory and field. J Econ Entomol 96:1031–1038

    Article  PubMed  CAS  Google Scholar 

  • Trolinder NL, Goodin JR (1987) Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.). Plant Cell Rep 6:231–234

    Article  CAS  Google Scholar 

  • Wu JH, Zhang XL, Nie YC, Luo XL (2005) High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants. Plant Breed 124:142–146

    Article  CAS  Google Scholar 

  • Yu CG, Mullins MA, Warren GW, Koziel MG, Estruch JJ (1997) The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects. Appl Environ Microbiol 63:532–536

    PubMed  CAS  Google Scholar 

  • Zhao JZ, Li YX, Collins HL, Shelton AM (2002) Examination of the F2 screen for rare resistance alleles to Bacillus thuringiensis toxins in the diamondback moth (Lepidoptera: Plutellidae). J Econ Entomol 95:14–21

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This research was funded by the National Program on Research and Development of Transgenic Plants, the Pilot Project of Chinese Academy of Sciences and National Special Project of Agricultural Public Sector. The authors thank Prof. Khizar Hayat Bhatti at the University of Gujrat (Pakistan) for many helpful suggestions for revising the manuscript.

Author information

Authors and Affiliations

  1. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, People’s Republic of China

    Jiahe Wu, Xiangrong Zhang & Yingchuan Tian

  2. Institute of Cotton Research, Shanxi Academy of Agricultural Sciences, 044000, Yuncheng, People’s Republic of China

    Xiaoli Luo & Yuejing Shi

Authors
  1. Jiahe Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoli Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiangrong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuejing Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yingchuan Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jiahe Wu or Yingchuan Tian.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11248_2011_9483_MOESM1_ESM.tif

Supplementary material 1 (TIFF 210 kb) Fig. S1 Schematic diagram of the T-DNA structure of two expression vectors, pBVip3A* (A) and pBTVip3A* (B) NPT II, neomycin phosphotransferase gene; DE-35SP, CaMV35S promoter with double enhancer sequence; Ω, the fragment of TMV-RNA cDNA; Nos T, transcriptional termination sequence of nopaline synthase gene; LB, left border of T-DNA; RB, right border of T-DNA

11248_2011_9483_MOESM2_ESM.tif

Supplementary material 2 (TIFF 416 kb) Fig. S2 Molecular identification of transgenic plants A: PCR analysis of transgenic cotton plants. Lane M: DNA ladder marker; lane 1: pBTVip3A* vector; lane 2 ~ 6: independent transgenic pBTVip3A* cotton plants; lane 7 ~ 11: independent transgenic pBVip3A* cotton plants; lane 12: non-transformed (NT) plant. B: Southern blot assay of vip3A* and tvip3A* transgenic cotton lines. The pBVip3A* plasmid and transformed cotton DNA were digested with Hind III (only one Hind III restriction site is present within the T-DNA region). The Vip3A*-specific probe was PCR-amplified from pBVip3A* vector. Plant DNAs are NT (non-transformed plant), three independent transgenic pBTVip3A* plants (CTV1, CTV2, and CTV3), and three independent pBVip3A* plants (CV1, CV2, and CV3). DNA markers are shown on the left side

11248_2011_9483_MOESM3_ESM.tif

Supplementary material 3 (TIFF 3304 kb) Fig. S3 Insect bioassays. A: The effects of the transgenic cotton lines on first-instar larvae of cotton bollworm (CBW, Helicoverpa zea) infested on the detached leaves. NT plants were used as negative controls. B: Mortalities of the larvae of fall armyworm (FBW, Spodoptera frugiperda), beet armyworm (BAW, Spodoptera exigua) and cotton bollworm CBW on the detached leaves from transgenic lines and NT plants in the greenhouse

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, J., Luo, X., Zhang, X. et al. Development of insect-resistant transgenic cotton with chimeric TVip3A* accumulating in chloroplasts. Transgenic Res 20, 963–973 (2011). https://doi.org/10.1007/s11248-011-9483-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-011-9483-0

Keywords

Search

Navigation