Amplification of the dreb2a transcription factor gene from salsola richteri (moq.) Kar. Ex litv. Growing in the southern Aralkum

American Journal Of Agriculture And Horticulture Innovations

28

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VOLUME

Vol.05 Issue03 2025

PAGE NO.

28-31

DOI

10.37547/ajahi/Volume05Issue03-08

Amplification of the dreb2a transcription factor gene
from salsola richteri (moq.) Kar. Ex litv. Growing in the
southern Aralkum

Doston Rizaev

Junior Researcher Institute of Bioorganic Chemistry of the Academy of Sciences of the Republic of Uzbekistan, Uzbekistan

Jamoliddin Ziyavitdinov

Doc. chem. sciences, prof. Institute of Bioorganic Chemistry of the Academy of Sciences of the Republic of Uzbekistan, Uzbekistan

Sanjar Sherimbetov

Doc. biol. sciences, prof. Institute of Bioorganic Chemistry of the Academy of Sciences of the Republic of Uzbekistan, Uzbekistan

Bahtiyor Adilov

Doc. biol. Sciences, Institute of Genetics and Experimental Plant Biology of the Academy of Sciences of the Republic of Uzbekistan,
Uzbekistan

Received:

29 January 2025;

Accepted:

28 February 2025;

Published:

31 March 2025

Abstract:

This study investigates halophytic plants of the genus Salsola L., which are widely spread in the southern

part of the Aralkum Desert. During the research, a PCR product of approximately 1200 bp associated with the
DREB2A gene was amplified in one of the species, Salsola richteri. This gene plays a key role in plant adaptation
to drought and salt stress. The obtained data will be used for sequencing the DREB2A gene and analyzing its
expression.

Keywords:

Salsola richteri, DREB2A, transcription factor, salt tolerant plants, primer.

Introduction:

Salsola richteri is a shrub or a small tree

ranging from 1 to 3 meters in height. In its young stage,
it is covered with finely tuberculate (or papillose)
leaves, which later become leafless. The stem is weakly
branched, with smooth gray bark, up to 5 cm thick at
the base, and its woody branches soon acquire a milky-
white color [1]. This species is promising for
phytoremediation practices due to its good growth on
sandy soils, high seed productivity, ability to propagate
by seeds and cuttings, and tolerance to significant
salinity. Its powerful root system makes it effective for
sand stabilization [4, 5]. The high protein content and
the formation of substantial organic mass with
economical water use allow S. richteri to be utilized as
a valuable forage plant for autumn-winter pastures [2,

3, 5].

Karakalpak scientists studied the growth and
development of S. richteri under the ecological
conditions of the Karakalpak part of the Kyzylkum
Desert from 2014 to 2018. Fruiting of S. richteri began
as early as the first year of life, in the third decade of
June. Fruits were mainly formed on fourth-order
shoots, with the number of fruits per shoot ranging
from 16 to 38 and the number of leaves from 14 to 40.
Fruiting depended on the age of the shrubs; in the
second year of cultivation, a single shrub could produce
between 3,000 and 6,000 seeds [5].

S. richteri belongs to the groups of euhalophytes and
hemixerophytes, which are adapted to soils with a
moderate level of salinity (1.8

–

2.3%; Cl 0.1

–

0.23%

—

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American Journal Of Agriculture And Horticulture Innovations (ISSN: 2771-2559)

chloride-sulfate and sulfate salinization or 1.3

–

1.8%; Cl

0.1

–

0.23%

—

sulfate-chloride and chloride salinization).

This species is capable of successfully growing in such
soils, maintaining its dominant position in the
phytocenosis even as salinity levels decrease. It forms
plant associations and formations over vast areas that
have been exposed after water receded. In his
research, S. G. Sherimbetov analyzed the chemical
composition of plants found in the desiccated regions
of the Aral Sea. Among the studied halophytes
(Climacoptera

aralensis,

Kalidium

capsicum,

Halostachys belangeriana, Salsola richteri, Haloxylon
aphyllum, Tamarix hispida), a high content of minerals
such as Ca, Cl, K, Mg, and Na was observed.

The plant Salsola drummondii Ulbr., belonging to the
genus Salsola L., is capable of successfully growing and
completing its life cycle under high salinity conditions
(500

–

800 mM NaCl). This plant adapts to salt stress by

reducing the level of photosynthetic pigments,
decreasing carotenoid content, and increasing the
activity of antioxidant enzymes [6]. Based on the genes
of Salsola iberica, which are responsible for resistance
to abiotic stress factors, a bank of expressed sequence
tags (EST

—

Expressed Sequence Tag) was created to

analyze the molecular mechanisms of adaptation. This
bank included 377 ESTs, which were grouped into 227
unique fragments. Similarities were found between S.
iberica ESTs and stress-resistance genes, including salt-
induced proteins, betaine-aldehyde dehydrogenases,
and calcium-binding proteins [7]. Salsola ferganica is a
desert herbaceous plant that grows in arid regions of
western and northwestern China.

To normalize gene expression in S. ferganica under
abiotic stress, nine reference genes (TUA-1726, TUA-
1760, TUB, GAPDH, ACT, 50S, HSC70, APT, and U-box)
were tested under six stress conditions. The analysis
revealed that ACT and U-box exhibited the highest
stability among all tested variants [8].

A review of scientific literature has shown that despite
S. richteri is tolerance to salinity and drought, there is a
lack of data on transcription factors responsible for
these adaptation mechanisms. Abiotic stress plays a
crucial role in plant growth and development, as plants
are exposed to various adverse factors such as drought,
low and high temperatures. Under stress conditions,
several stress-resistant genes are activated, among
which DREB (Dehydration Responsive Element Binding)
genes play a particularly important role. These genes
encode proteins of the Apetala2/ethylene (AP2/ERF)

family, which bind to the dehydration-responsive
element (DRE)/C-repeat in the promoter regions of
stress-resistance genes. The DRE cis-element, located
near the promoter regions of stress-associated genes,
serves as the binding site for DREB transcription
factors, which regulate osmotic stress in plants.
Drought and high salinity levels induce the expression
of the DREB2 gene, which plays a key role in regulating
abiotic stress-responsive genes.

The expression of OsDREB2A in Oryza sativa is
enhanced under salt stress and dehydration, but the
gene exhibits low sensitivity to low temperatures and
abscisic acid (ABA) [9]. Similarly, in Zea mays, the
transcript level of ZmDREB2A increases under high-
temperature stress. Arabidopsis thaliana demonstrates
DREB2A activation primarily in response to drought and
salt stress [10].

The aim of the study

is to design a new primer for

obtaining the full DREB2A gene sequence from S.
richteri, a plant from the Chenopodiaceae family.

METHODS

As research objects, plant biomaterials of S. richteri
collected from the Southern Aralkum in 2021 were
used. The plant materials were identified by staff of the
Institute of Botany, Academy of Sciences of the
Republic of Uzbekistan.

Total DNA was extracted from the leaves of the plant
using the PureLink Plant Total DNA Purification Kit
(Invitrogen by Thermo Fisher Scientific).

Primer Design

To amplify the DREB2A gene from plants of the
Chenopodiaceae family, a search for the nucleotide
sequence of the DREB2A gene was conducted in the
NCBI database (NCBI - www.ncbi.nlm.nih.gov) using the
BLASTN algorithm in the BLAST web application within
the GenBank database. Based on the nucleotide
sequences of the DREB2A gene from Salicornia
brachiata (ID: GU809211.1), Haloxylon ammodendron
(ID: KP765243.1), Beta vulgaris (ID: XM_010694618.3),
and Chenopodium quinoa (ID: XM_021876866.1) for
the reverse primer, as well as Chenopodium album (ID:
OX_419225.1) and Salicornia ramosissima (ID:
OX_596239.1) for the forward primer from the NCBI
database, a design of specific degenerate primers was
created for the amplification of approximately 900 and
1500 nucleotide sequences using the CLUSTAL O(1.2.4)
Multiple Sequence Alignment program (Table 1).

Table 1.

Oligonucleotide sequence of primers for amplification of the DREB2A gene of some

species of the

Chenopodiaceae

family

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American Journal Of Agriculture And Horticulture Innovations (ISSN: 2771-2559)

Polymerase chain reaction

The amplification of DREB2A gene fragments from the
studied halophytic plant Salsola richteri was performed

using a MiniAmp™ Plus Thermal Cycler (Applied

BioSystems, USA) with specific primers. The
polymerase chain reaction (PCR) of the DREB2A gene
was carried out according to the protocol of the

Phusion™ High

-Fidelity DNA Polymerase kit (Thermo

Fisher

Scientific,

USA) [7].

For

the

quantitative determination of nucleotides in the PCR
products, electrophoresis was performed in a 2%
agarose gel. The length of the PCR products was
determined using a 100 bp DNA Ladder marker
(Invitrogen, USA) (Fig. 1).

Figure 1. Electropherogram of the PCR product of the DREB2A gene

M - DNA marker; 1. S. richteri; 2. S. richteri

RESULTS AND DISCUSSION

As a result of the study, a PCR product with an
approximate length of 900 and 1200 base pairs was
obtained from Salsola richteri. It was established that
the PCR product length corresponds to the predicted
size during primer design, confirming their specific
binding to the complementary sequence. According to
NCBI, the length of the DREB2A gene in the registered
species Salicornia brachiata (ID: GU809211.1),
Haloxylon ammodendron (ID: KP765243.1), Beta
vulgaris (ID: XM_010694618.3), and Chenopodium
quinoa (ID: XM_021876866.1) ranges from 1100 to
1200 base pairs. The length of the PCR products
obtained from S. richteri also falls within this range,
confirming their affiliation with the DREB2A gene. The
obtained results enable further investigation of the
nucleotide sequence of the DREB2A gene in S. richteri

and the study of its expression levels under drought
and salinity conditions.

CONCLUSION

DNA fragments were isolated from plants of the
Chenopodiaceae family distributed in the Southern
Aralkum. Using bioinformatics online resources,
specific DREB2A primers were designed. The DREB2A
gene was successfully amplified from the DNA of
Salsola richteri using PCR. It was established that the
PCR products obtained from S. richteri were suitable
for sequencing nucleotide pair sequences. The results
of this study provide a foundation for further analytical
research aimed at understanding the functionality of
the DREB2A gene in S. richteri.

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5

'

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'

Oligonucleotide sequence

Pr_D_F

TCGAAGAAAGGDTGTATGAAAGG

Pr_Dr_F1

GGGAWRTTTTAWAWWTTKATTTA

Pr-DOST_R1

AAACCTAYWGAGAATAAGCTT

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