1022-7954/03/3911- $25.00 © 2003
Russian Journal of Genetics, Vol. 39, No. 11, 2003, pp. 1258–1264. Translated from Genetika, Vol. 39, No. 11, 2003, pp. 1490–1497.
Original Russian Text Copyright © 2003 by Speransky, Dorokhin, Novikova, Shevelev, Valueva.
Potato tubers contain a large group of soluble pro-
teins, including proteinase inhibitors of different
classes . Among these are several 20–24-kDa pro-
teins belonging to the family of the soybean Kunitz
trypsin inhibitors (SKTI). The potato proteinase inhib-
itors exhibit homology to the soybean Kunitz protein-
ase inhibitors, and are classiﬁed into the subfamily of
potato Kunitz proteinase inhibitors (PKPIs). Physiolog-
ical role played by these inhibitors in plants as well as
the pathways of their in vivo expression still remain
unclear. It is suggested that PKPIs can be involved in
the defense response of a host plant against the action
of phytopathogen by inhibiting the activity of phyto-
pathogenic proteinases [2–4].
Different isoforms of these proteins are probably
encoded by a family of closely related genes [5, 6].
Currently, based on the analysis of nucleotide and
amino acid sequences, all proteins of the PKPIs sub-
family and the corresponding genes are subdivided into
three major homology groups: PKPI-A, -B, and -C .
To date, more than 30 individual nucleotide sequences
(mostly cDNA), encoding PKPIs from different potato
cultivars have been described. A great number of the
PKPIs sequences, both cDNA and genomic, are depos-
ited in the NCBI database, but not yet published.
In the present study we have shown that the potato
genome of cv. Istrinskii contains a gene with yet
unknown sequence, which encodes the PSPI-21-6.3
protein, isolated and described earlier from the tuberous
juice of the same cultivar [7–9]. Natural PSPI-21-6.3 pro-
tein is the human leukocyte elastase inhibitor, which
consists of two chains, A and B, (with total molecular
weight of 21 kDa) connected by a disulﬁde bridge .
Based on the analysis of its amino acid sequence, this
protein should be attributed to the group PKPI-B.
In addition to the protein mentioned, tuberous juice
of this potato cultivar also contains a number of other
proteins belonging to the PKPI subfamily. One of these
proteins, PSPI-21-5.2, was characterized in great detail
[7, 9]. This protein represents an isoform exhibiting
homology to the PSPI-21-6.3, with analogous molecu-
lar weight and speciﬁcity. Two other proteins, PSPI-22
and PCPI-23, with molecular weights of 22 and 23 kDa,
respectively, were partly characterized .
The present study was focused on cloning and deter-
mination of the DNA sequence encoding the PKPIs
from potato genome of cv. Istrinskii.
MATERIALS AND METHODS
The experiments were carried out using the potato
L., cv. Istrinskii). Tubers
were kept in dark at +4
C for 2–4 months. After that
they were sprouted in a humid chamber at the daylight
illumination and room temperature for 2 weeks. The
young sprouts were used for DNA isolation as
described in .
strain BMH 78-18 (
United States) was used to maintain vectors pUK21 
and pGEM-T-easy (Promega, United States).
Primers KEN1 5'(GGGAGCTCAACCTAC-
CTAGTGATGCTACT)3' and KEN2 5'(GGGTCGA-
CATTTGTTATTACT)3' were constructed with regard
to the PKPIs gene sequences reported in .
Molecular cloning, plasmid DNA isolation, and
digestion with restriction endonucleases were carried
out as described in .
Cloning of a Sequence
Encoding the SKTI Family Proteinase Inhibitor from Potato
A. S. Speransky
, A. V. Dorokhin
, S. I. Novikova
A. B. Shevelev
, and T. A. Valueva
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia; e-mail: firstname.lastname@example.org
Russian State Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 113545 Russia
Received December 10, 2002; in ﬁnal form, April 30, 2003
—Seven structurally similar clones from potato (
L.), cv. Istrinskii genomic DNA
were isolated by cloning of the PCR products. It was suggested that ﬁve of these clones were the ampliﬁed cop-
ies of the same gene. Based on comparative and structural analysis of these clones, initial nucleotide structure
of the gene was reconstructed. It appeared to be highly homologous (98%) to the already published sequences
encoding the proteins belonging to the soybean Kunitz trypsin inhibitor family (SKTI). Comparison of the
results with the previously published data on the SKTI-type proteinase inhibitors from potato of cv. Istrinskii
suggests that the gene examined encodes both chains of the earlier described PSPI-21-6.3 protein .