1022-7954/05/4102- © 2005 Pleiades Publishing, Inc.
Russian Journal of Genetics, Vol. 41, No. 2, 2005, pp. 112–115. Translated from Genetika, Vol. 41, No. 2, 2005, pp. 166–170.
Original Russian Text Copyright © 2005 by Ogarkova, Tomilov, Tomilova, Pogorelko, Tarasov.
In recent years, the structure and function of the
higher plant genome, in particular, of
, has been often studied using insertion
mutagenesis. In many laboratories over the world,
expanded collections of insertion mutants have been
developed [1–5]. In 2000, the complete genome of
has been sequenced; the results are avail-
able in the computer databases at [http//kazusa.or.jp/,
http//www.ncbi.nlm.nih.gov/]. In view of this, the strat-
egy of studying the structure and function of
genome has altered. Based on the computer databases,
a gene can be identiﬁed from a small DNA sequence
near insertion. Using this approach, we identiﬁed the
gene, an insertion into which resulted in changes in
MATERIALS AND METHODS
We used the collection of inser-
tion morphological mutants of
Köln, which was obtained by agrobacterial transforma-
tion of germinating seeds by the binary vector system
Previously, we described in detail the media, the
cultivation conditions, selection of transformed plants
and testing for their transgenic nature, histochemical
analysis of the reporter gene of
isolation and analysis of DNA from plant tissues, con-
ditions and primers for PCR used to detect the trans-
formed plant T-DNA, and determining the number of
T-DNA insertions .
Molecular biological analysis of the insertion
DNA molecular cloning and sequencing were
conducted using standard procedures. The genomic
DNA region adjacent to T-DNA insertion was ampliﬁed
in TAIL-PCR, in which random degenerated primers
participated in the reaction along with three successive
primers speciﬁc for the left border of T-DNA regions
. The integration sites of T-DNA insertions in the
genome of line 176 were determined in PCR with the
following pairs of primers:
chromosome 2; 5'-GAAATGCCTCGTGTCTGCCT-
CAAAG-3' and 5'-AAACCCAACTAATAAATACTC-
TACT-3' for chromosome 3.
Analysis for phenotype segregation.
phenotypes were determined in generations T3 and T4
in successive experimental series.
Rescue of lethal seedlings
was performed using
exogenous phytohormones .
RESULTS AND DISCUSSION
Isolation and characterization of the mutant.
recessive and lethal mutant of the
is represented by line 176 in the previously developed
After cocultivation with agrobacteria, the ﬁrst gen-
eration seeds (T1) were germinated aseptically in soil.
The transgenic plant selection in the following genera-
tion (T2) passed through several stages . As a result,
a kanamycin-resistant plant with normal morphology
was obtained, the cells of which exhibited GUS activ-
ity, whereas PCR analysis conﬁrmed the presence of T-
Identification of the Gene Whose Mutation Leads
to the Morphological Changes in the Hypocotyl
O. A. Ogarkova, A. A. Tomilov, N. B. Tomilova, G. V. Pogorelko, and V. A. Tarasov
Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia
Fax: (095)135-43-04; e-mail: firstname.lastname@example.org
Received July 13, 2004
—The results of genetic and molecular genetic analysis of line 176 of
reduced hypocotyls obtained from a previously developed collection of insertion mutants, are presented. The
examined mutation proved to be recessive and based on a single insertion of the T-DNA vector pLD3 into the
genome. Computer-aided analysis of the ampliﬁed in TAIL–RCR DNA region adjacent to the left
border of the insertion revealed a putative site of T-DNA insertion, the 2.5-kb
gene located in the
long arm of chromosome 2, near the centromere.