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C E L L CU LTUR E F O R PRODUCING PLA N T S R E SISTA N T T O BIOTIC AND AB IO TIC
FACTOR S
APPROACHES TO CLONINGOFSPECIFICGENES INVOLVED INTO
PLANTRESPONSE UNDER STRESS CONDITIONS
I.O.Vakulenko, A.O.Golovko, Y.I.Ratushnyak, V.A.Rudas, K.S.Sytnik,
N.V.Kuchuk, Abul Mandal*, Tapio Palva**
Institute o fCell Biology and GeneticEngeneering, Academy o fSciences Ukraine, 252143,
Kiev 143, Ulitza Zabolotnogo, 148.E-Mail:
*Swedish University o fAgricultural Sciences, Uppsala, Sweden.
**University o fHelsinki, Helsinki, Finland
The goal o f this work is identification and cloning o f specific genes
involved into plant response under chilling and oxidative stress conditions.
To create the collection o f transformants insertional mutagenesis method
was used. This method is based on ability o f Agrobacterium tumerfaciens
to transfe rthe segments o f DNA(T-DNA) to plant cells where they integrate
into nuclear chromosomes.
Two tagging vectors have been used in our experiments. The first one
(pMHA2) har-boured a promoterless GUS reporter gene placed adjacent to
the right border o f the T-DNA. It also harboured the pnos-nptll- pAnos gene
to be used fo r selection o f transformants. The second one (pHTT654/
pGV2260) incorporated GFP gene as a reporter gene. It can be suggested
that a promoterless reporter gene will be controlled by plant promotor elements
in case the genetic transformation occurs. Arabidopsis thaliana ecotype
C24 was chosen as a model object for the transformation. The second
generation oftransformed plants was undergone chilling or incorporation the
special herbicide paraquat which was known to induce oxidative stress in
plants. GUS-GFP activity was studied after that treatment. 644 and 100
transgenic lines have been produced using pMHA2 and pHTT654/pGV2260.
Second generation of 644 lines was screened and 220 lines were collected
because of their ability to grow in the presence of kanamycin. 113 lines have
been tested. Two trangenic lines have been identified in which expression of
GUS reporter was induced by treatment with paraquat, and one has been
identified in which expression o f GUS reporter was induced under chilling.
Transgenic lines obtained will be used as a starting material fo r cloning o f
the plant promoter sequences and genes induced by stress conditions.
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