Transfer and Expression of an Artificial Storage Protein (ASP1) Gene in Cassava (Manihot Esculenta Crantz)

In order to increase the nutritional quality of cassava storage roots, which contain up to 85% starch of their dry weight, but are deficient in protein, a synthetic ASP1 gene encoding a storage protein rich in essential amino acids (80%) was introduced into embryogenic suspensions of cassava via Agrobacterium-mediated gene transfer. Transgenic plants were regenerated from suspension lines derived from hygromycin-resistant friable embryogenic callus lines. Molecular analysis showed the stable integration of asp1 in cassava genome and its expression at RNA level in transformed suspension lines. PCR and Southern analyses proved the transgenic nature of the regenerated plant lines. The expression of asp1 at RNA level was demonstrated by RT-PCR. The ASP1 tetramer could be detected in leaves as well as in primary roots of cultured transgenic plants by western blots. These results indicate that the nutritional improvement of cassava storage roots may be achieved by constitutive expression of asp1 in transgenic plants.

[1]  P Argos,et al.  A structural model for maize zein proteins. , 2004, The Journal of biological chemistry.

[2]  A. Aryan,et al.  Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.) , 1996, Transgenic Research.

[3]  J. Murray,et al.  Isolation of intact DNA and RNA from plant tissues. , 1994, Analytical biochemistry.

[4]  K. W. Pearson,et al.  Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine , 2004, Plant Molecular Biology.

[5]  Claude Fauquet,et al.  Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures , 1996, Nature Biotechnology.

[6]  J. Puonti-Kaerlas,et al.  PIG-mediated cassava transformation using positive and negative selection , 2000, Plant Cell Reports.

[7]  G. Galili.  Regulation of Lysine and Threonine Synthesis. , 1995, The Plant cell.

[8]  E. Sofiari,et al.  Production of transgenic cassava (Manihot esculenta Crantz) plants by particle bombardment using luciferase activity as selection marker , 1996, Molecular Breeding.

[9]  J. Jaynes,et al.  Enhancing the Nutritional Quality of Crop Plants: Design, Construction, and Expression of an Artificial Plant Storage Protein Gene , 1992 .

[10]  I. Potrykus,et al.  Efficient Production of Transgenic Cassava Using Negative and Positive Selection , 2000, Transgenic Research.

[11]  G. Gómez,et al.  The amino acid composition of cassava leaves, foliage, root tissues and whole-root chips , 1986 .

[12]  J. Puonti-Kaerlas,et al.  Production of stably transformed cassava plants via particle bombardment , 2000, Plant Cell Reports.

[13]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[14]  J. Cock.  Cassava: New Potential For A Neglected Crop , 1984 .

[15]  I. Potrykus,et al.  Regeneration of cassava plants via shoot organogenesis , 1998, Plant Cell Reports.

[16]  M. Montagu,et al.  Enkephalins Prouduced in Transgenic Plants Using Modified 2S Seed Storage Proteins , 1989, Bio/Technology.

[17]  F. White,et al.  Expression of a synthetic gene for improved protein quality in transformed potato plants , 1989 .

[18]  R. U. Schenk,et al.  Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures , 1972 .

[19]  G. Galili,et al.  Seed-specific expression of a bacterial desensitized aspartate kinase increases the production of seed threonine and methionine in transgenic tobacco , 1993 .

[20]  F. Skoog,et al.  A revised medium for the growth and bioassay with tobacco tissue culture , 1962 .

[21]  C. Fauquet,et al.  Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures , 1998, Plant Cell Reports.

[22]  Nigel J. Taylor,et al.  Development of friable embryogenic callus and embryogenic suspension culture systems in cassava (Manihot esculenta Crantz) , 1996, Nature Biotechnology.

[23]  Shashi Sharma,et al.  Expression of a sulfur-rich maize seed storage protein, δ-zein, in white clover (shape Trifolium repens) to improve forage quality , 1998, Molecular Breeding.

[24]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[25]  P. Hirsch,et al.  A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid , 1983, Nature.

[26]  M. Van Montagu,et al.  Stable Accumulation of Modified 2S Albumin Seed Storage Proteins with Higher Methionine Contents in Transgenic Plants. , 1990, Plant physiology.

[27]  J. Morrissey,et al.  Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. , 1981, Analytical biochemistry.

[28]  O. Anderson,et al.  Expression of a novel high-molecular-weight glutenin subunit gene in transgenic wheat , 1996, Nature Biotechnology.

[29]  B. Larkins,et al.  Expression of a mutant alpha-zein creates the floury2 phenotype in transgenic maize. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[30]  D. Ludevid,et al.  Lysine-rich c-zeins are secreted in transgenic Arabidopsis plants , 1998, Planta.

[31]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[32]  Ingo Potrykus,et al.  Genetic transformation of cassava (Manihot esculenta Crantz) , 1996, Nature Biotechnology.

[33]  H. Katinger,et al.  Efficient transformation of Agrobacterium spp. by electroporation. , 1989, Nucleic acids research.

[34]  V. Srivastava,et al.  Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat , 1996, Nature Biotechnology.

[35]  J. Kemp,et al.  A modified 10 kD zein protein produces two morphologically distinct protein bodies in transgenic tobacco , 2000 .

[36]  B. Larkins,et al.  The maize gamma-zein sequesters alpha-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm. , 1996, The Plant cell.