A phytoene synthase (psy) open reading frame from β-carotene-rich sweet potato as an
alternative gene for cassava genetic transformation.
Parra S1, Toro N2, Beltran JA1, Chavarriaga P 1, and Tohme J1
1. Conservation and Use of Tropical Genetic Resources, CIAT, AA 6713 , Cali, Colombia.
2. Universidad del Valle, Departamento de Biología, Cali, Colombia.
INTRODUCTION • 5’ RACE
By means of genetic transformation we seek to increase the β-carotene
content in cassava roots by over-expressing, in a tissue-specific With the first amplification towrads the cDNA 5’ end we obtained a
manner, genes of the carotenoid synthesis pathway. Currently we are band of approximately 800bp (Figure 2A). It was isolated and
using the bacterial gene crtB as a source of Phytoene Synthase (psy), sequenced. The 783bp obtained was similar (E-value = 0) to the C.
which has proven to be efficient to enhance carotenoid production in canephora psy sequence. The comparison between the sequence
fruits, seeds and roots of several crops obtained and other psy sequence showed a lack of a transit peptide.
Therefore we performed a second amplification towards the cDNA
However, plant derived genes, besides having the advantage of being 5’end and obtained an extra 200bp (Figure 2B) product. After
better accepted by the public, may be a more efficient source of PSY, characterizing the product, 143 nucleotides were confirmed to be
as it has been proven with Golden Rice. We are following the same psy sequence, with similarity (E-value = 6.3x10-23) to the L.
approach for cassava and therefore have cloned the psy gene sequence esculentum psy gene.
from β-carotene rich sweet potato storage roots (Ipomoea batatas) by
means of rapid amplification of cDNA ends (RACE).
• Primers design
Figure 2. Amplification results to the 5’cDNA end. A. First amplification.
B. Second amplification.
Degenerate forward primers were designed considering the alignment
result of psy sequences from Coffea canephora (Robusta Coffee),
Gentiana lutea (Gentian), Lycium barbarum (Goji), Capsicum annuum With the three different sequences of the cDNA ends we predicted a
(Pepper) and Nicotiana langsdorffii (Tobacco), to amplify the 3’ cDNA contig and suggested an open reading frame (ORF) for the sweet
end. With the known sequences obtained after 3’ end amplification potato psy gene.
and characterization, we designed specific reverse primers to
synthesize the cDNA and amplify the 5’ end.
• Complete psy ORF isolation
• Sequence isolation Using a pair of specific primers we isolated the complete psy ORF.
The amplification product had 1000bp (Figure 3A) approximately,
Single strand cDNA with
which was isolated and characterized. We obtained a 1029bp open
adapter ccc reading frame that has a predicted protein of 342 aminoacids. The
comparison with the Swissprot database showed high similarity (E-
value = 3.9x10-176) with the enzyme PSY of L. esculentum (Figure
Specific primers design
Amplification with outer
3’cDNA sequence Specific primers
Figure 3. Sweet potato psy ORF. A. Amplification result with specific primers. B.
sequence Alignment result between the predicted aminoacids sequences and others reported in
Nested amplification with the Swissprot database.
RESULTS AND DISCUSSION PERSPECTIVES
• 3’ RACE
• Cloning the psy ORF in an expression vector under a cassava root-
A product of approximately 500bp (Fig 1) was obtained with the specific promoter, or a constitutive promoter, to transform cassava
degenerate primers in the 3’cDNA end nested amplification. Then, embryogenic cells, to verify that the ORF produces a functional
this product was isolated from the gel and sequenced. The 480bp enzyme.
sequence was similar (E-value = 4.8x10-88) to the Lycopersicon
esculentum (tomato) psy gene. • Cloning the promoter of the psy sequence to use it as an alternative
source of regulatory regions for the expression of carotenogenic genes
in cassava roots.
Figure 1. Nested amplification result to the 3’cDNA end
Date prepared: July 2008