3 Biotech

, 9:139 | Cite as

Genetic transformation of Chlorella vulgaris mediated by HIV-TAT peptide

  • Pavan GadamchettyEmail author
  • Phanindra Lakshmi Venkata Mullapudi
  • Raghavendrarao Sanagala
  • Manickavasagam Markandan
  • Ananda Kumar Polumetla
Original Article


Scientific interest in microalgal species is growing and, genetic transformation has definitely opened more avenues, in the ongoing research on microphytes. In the present study, we have attempted to transform Chlorella vulgaris by mobilizing double-stranded linear Transfer DNA (T-DNA) comprised of green fluorescent protein (egfp) gene cassette and hygromycin phosphotransferase II (hptII) gene cassette non-covalently bound to TAT peptide, into C. vulgaris cells treated with Triton X-100. The transformed C. vulgaris cells when examined under fluorescent microscope, exhibited green fluorescence in comparison to the untransformed cells. The transformed cells were further screened, and the surviving colonies were sub-cultured, on BG11 medium fortified with Hygromycin. The surviving colonies were confirmed for the presence of integrated T-DNA by Polymerase Chain Reaction with egfp and hptII gene-specific primers. This methodology has potential to substitute the existing tedious transformation methodologies and ease the future studies in microalgae.


Chlorella vulgaris HIV-TAT Cell-penetrating peptide Genetic transformation Microalgae 



The authors thankfully acknowledge the support from The Centre for Conservation and Utilization of Blue Green Algae (CCUBGA), Division of Microbiology, IARI, New Delhi and technical staff at NRCPB, IARI, New Delhi.

Author contributions

The experiments were conceived and designed by PAK, MLVP and GP. The experiments were performed by GP and SR. The data were analyzed by MM and SR. The reagents, materials and analysis tools were provided by PAK. The manuscript was prepared by GP and MLVP.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.


  1. Cha TS, Yee W, Azia A (2012) Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris. World J Microbiol Biotechnol 28:1771–1779CrossRefGoogle Scholar
  2. Chen CP, Chou JC, Liu BR, Chang M, Lee HJ (2007) Transfection and expression of plasmid DNA in plant cells by arginine-rich intracellular delivery peptide without protoplast preparation. FEBS Lett 581:1891–1897CrossRefGoogle Scholar
  3. Chow KC, Tung WL (1999) Electrotransformation of Chlorella vulgaris. Plant Cell Rep 18:778–780CrossRefGoogle Scholar
  4. Chuah JA, Yoshizumi T, Kodama Y, Numata K (2015) Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers. Sci Rep 5:7751CrossRefGoogle Scholar
  5. Chugh A, Eudes F (2007) Translocation and nuclear accumulation of monomer and dimer of HIV-1Tat basic domain in triticale mesophyll protoplasts. Biochim Biophys Acta 1768:419–426CrossRefGoogle Scholar
  6. Chugh A, Amundsen E, Eudes F (2009) Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores. Plant Cell Rep 28:801–810CrossRefGoogle Scholar
  7. Converti A, Casazza AA, Ortiz EY (2009) Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem Eng Process 48(6):1146–1151CrossRefGoogle Scholar
  8. Corre G, Templier J, Largeau C, Rousseau B, Berkalo C (1996) Influence of cell wall composition on the resistance of two Chlorella species (Chlorophyta) to detergents. J Phycol 32:584–590CrossRefGoogle Scholar
  9. Dawson HN, Burlingame R, Cannons AC (1997) Stable transformation of Chlorella: rescue of nitrate reductase-deficient mutants with the nitrate reductase gene. Curr Microbiol 35:356–362CrossRefGoogle Scholar
  10. El-Sheekh MM (1999) Stable transformation of the intact cells of Chlorella kessleri with high velocity microprojectiles. Biol Plant 42:209–216CrossRefGoogle Scholar
  11. Gomma AE, Lee SK, Sun SM, Yang SH, Chung G (2015) Development of stable marker-free nuclear transformation strategy in the green microalga Chlorella vulgaris. Afr J Biotechnol 14(37):2715–2723CrossRefGoogle Scholar
  12. Gump JM, Dowdy SF (2007) TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med 13(10):443–448CrossRefGoogle Scholar
  13. Hyman JM, Geihe EI, Trantow BM, Parvin B, Wender PA (2012) A molecular method for the delivery of small molecules and proteins across the cell wall of algae using molecular transporters. Proc Natl Acad Sci USA 109:13225–13230CrossRefGoogle Scholar
  14. Kang S, Suresha A, Kim YC (2017) A highly efficient cell penetrating peptide pVEC-mediated protein delivery system into microalgae. Algal Res 24:360–367CrossRefGoogle Scholar
  15. Kim D, Kim YT, Cho JJ, Bae J, Hur S, Hwang I, Choi T (2002) Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea. Mar Biotechnol 4:63–73CrossRefGoogle Scholar
  16. Kim HH, Lee WS, Yang JM, Shin S (2003) Basic peptide system for efficient delivery of foreign genes. Biochim Biophys Acta 1640:129–136CrossRefGoogle Scholar
  17. Koley D, Bard AJ (2010) Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM). Proc Natl Acad Sci USA 107:16783–16787CrossRefGoogle Scholar
  18. Kumar M, Jeon J, Choi J, Kim SR (2018) Rapid and efficient genetic transformation of the green microalga Chlorella vulgaris. J Appl Phycol 30(3):1735–1745CrossRefGoogle Scholar
  19. Liu BR, Chou J-C, Lee H-J (2008) Cell membrane diversity in noncovalent protein transduction. J Membr Biol 222:1–15CrossRefGoogle Scholar
  20. Liu BR, Huang YW, Lee HJ (2013) Mechanistic studies of intracellular delivery of proteins by cell-penetrating peptides in cyanobacteria. BMC Microbiol 13(1):57CrossRefGoogle Scholar
  21. Niu YF, Zhang MH, Xie WH, Li JN, Gao YF, Yang WD, Liu JS, Li HY (2011) A new inducible expression system in a transformed green alga, Chlorella vulgaris. Genetic Mol Res 10(4):3427–3434CrossRefGoogle Scholar
  22. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  23. Sambrook J, Fritschi EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  24. Suresh A, Kim YC (2013) Translocation of cell penetrating peptides on Chlamydomonas reinhardtii. Biotechnol Bioeng 110:2795–2801CrossRefGoogle Scholar
  25. Tear CJ, Lim C, Wu J, Zhao H (2013) Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris. Microb Cell Factories 12:106CrossRefGoogle Scholar
  26. Watts A, Singh SK, Bhadouria J, Naresh V, Bishoyi AK, Geetha KA, Chamola R et al (2017) Brassica juncea lines with substituted chimeric GFPCENH3 give haploid and aneuploid progenies on crossing with other lines. Front Plant Sci 7:2019CrossRefGoogle Scholar
  27. Wei Y, Niu J, Huan L, Huang A, He L, Wang G (2015) Cell penetrating peptide can transport dsRNA into microalgae with thin cell walls. Algal Res 8:135–139CrossRefGoogle Scholar
  28. Yang B, Liu J, Liu B, Sun P, Ma X, Jiang Y et al (2015) Development of a stable genetic system for Chlorella vulgaris—a promising green alga for CO2 biomitigation. Algal Res 12:134–141CrossRefGoogle Scholar
  29. Ziemienowicz A, Tzfira T, Hohn B (2008) Mechanisms of T-DNA integration. Agrobacterium: from biology to biotechnology. Springer, New York, pp 396–441Google Scholar
  30. Ziemienowicz A, Shim YS, Matsuoka A, Eudes F, Kovalchuk I (2012) A novel method of transgene delivery into triticale plants using the Agrobacterium transferred DNA-derived nano complex. Plant Physiol 158:1503–1513CrossRefGoogle Scholar
  31. Zou L, Peng Q, Wang P, Zhou B (2017) Progress in research and application of HIV-1 TAT derived cell-penetrating peptide. J Membr Biol 250(2):115–122CrossRefGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.National Research Centre on Plant BiotechnologyNew DelhiIndia
  2. 2.Department of Biotechnology and Genetic Engineering, School of BiotechnologyBharathidasan UniversityTiruchirappalliIndia
  3. 3.Visargha Agri Sciences Private LimitedBhubaneswarIndia
  4. 4.Ganga Kaveri Seeds Private LimitedHyderabadIndia
  5. 5.Indian Institute of Rice ResearchHyderabadIndia

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