Biological and Clinical Sciences Research Journal
ISSN:
2708-2261
www.bcsrj.com
DOI:
https://doi.org/10.47264/bcsrj0201002
Biol. Clin.
Sci. Res. J., Volume, 2021: e002
Original Research Article
CLONING
AND EXPRESSION OF UNIVERSAL STRESS PROTEIN 2 (USP2) GENE IN ESCHERICHIA COLI
AKRAM A1, ARSHAD K1, HAFEEZ MN2
1Institute
of Molecular Biology and Biotechnology, University of Lahore, Lahore, Pakistan
2Department of Pharmacy, University of Chieti –
Pescara “G. d’Annunzio”, Chieti, Italy
Corresponding author email: kanzaarshad267@gmail.com,
chnadeemhfz@gmail.com
Abstract
Different types of abiotic stresses inhibit the normal growth
of plants by changing their physical biochemical, morphological, and molecular
traits. It links to the polygenic traits, which is controlled with the help of
different genes, due to this polygenetic the manipulation of foreign genetic
makeup is very difficult. Drought stress is the very major type of threat to
reduce the yield of cash crops in Pakistan and as well as in all over the
world. Gene manipulation is the solution to face this problem by producing
genetically modified crop plants that have the ability to survive in drought
conditions. Universal stress protein gene has been already identified in
bacteria which showed its response under stressed conditions, by manipulation
of universal stress protein gene. It was found from our study that the
bacterial cells transformed with the USP2 gene isolated from cotton induced
abiotic stress tolerance under heat, osmotic, and salt stress. It was suggested
from our findings that the USP2 gene could be used to produce abiotic stress
tolerance transgenic crop plants to enhance crop plant yield and quality.
Keywords: drought, heat, salt, universal stress protein gene, gene manipulation, polygenic
traits, tress tolerance
Introduction
There are many abiotic stresses which inhibt the normal
growth of plant. These unfavorable conditions stop the growth of crops, some
abiotic factors are drought tolerance, temperature flactuation, higher
concentration of salt, and cold stress (Jaleel et al., 2009). Loss of water at normal or higher temperature is known as drought cause
abnormalities in biological and chemical functions and also change the
morphology of cells, due to loss of water plant loses their turger pressure.
the composition of cell membrane is also changed and its interections between
all other components. Plants shows their response against drought stress at
moleculer and cellular level by activating the set of metabolic activities (Fahramand et al., 2014). At moleculer level, set of different genes are involved to start the
defence machanism of plant to servive under stres conditions, but the study of
genetic material of plant at proteomic and transcriptomic level shows that regulation of stress related genes are at
cellular level. It also shows that the
expression of gens are vary plant to plant (Ribas et al., 2006).
The activation of the metabolic reaction in stress condiotion
depends upon the two types of pathway known as ABA-dependent or ABA-independent
pathway,the end product of these pathways played an important role in the
activation of stress related pathway (Majid et al., 2017; Qamar et al., 2017; Shinozaki and Yamaguchi-Shinozaki,
2007). Abiotic stresses depends upon the
biological processes of plant. The
Plants contain bundle of protein contaning universal stress protein
domains. The study of plant taxa shows
200 different protein containing USP domains (Isokpehi et al., 2011; Sivamani et al., 2000). In cotton two genes are isolated
which encode universal stress protein GUSP1 and GUSP2 (Fahramand et al., 2014; Mahajan and Tuteja, 2005; Wang et al., 2003). The number of copies of genes
increased during drought stress. The role of universal stress protein first
studied in tomato shows that two genes
has been in the activtion of USP
protein. One gene produced light harvesting chlorophyll ‘a’ and ‘b’ binding
protein (LHCB) and second produced osmo protective compound which is proline (Fahramand et al., 2014). Some proteins are also discovered which played an important role to
prevent plant cells from cell to lose water (dehydration) (Van Loon and Van Lanen, 2013;
Van Loon et al., 2016). Universal stress proetin is present in cytoplasm also known as
cytoplasmic protein presenrt in bacteria. By chinging the concentration of
cellular components with the help of different types of abiotic stresses increased the expression of universal stress
protein (Jaleel et al., 2009). This group of universal stress protein is also present in different
types of bacteria, archaea, fungi, and plants. Six universal stress proteins
(USPa, USPb, USPc, USPd, USPe, USPf)
present in E. coli (Robinson, 2016) are further divide into two groups on the basis of
their sequence (Miki and McHugh, 2004; Tranel and Wright, 2002; Zhu et al., 2000). Universal stress protein plays an
important role in response to heat
shocks, cold shock and metabolic control (Adugna et al., 2006; Deng et al., 2011). Universal stress protein contain USP specfic domain that’s why it named
as universal stress protein(USP). A universal protein contain more then one USP
domains due to which it performs variable functions in different organisms.
With the help of 2-D-gel electrophoresis
first universal stress protein was discovered named as C-13.5 name based on its
migration during experiment. In further different studies role of universal stress protein is
identified. Universal stress protein play different role in cell to prevent
heat cold and from drought (Hanin et al., 2001; Lloyd et al., 2005). In drought stress universal stress protein shows intraction with annexin
protein but the detailed mechnism is still unknown. Under high salt conditions
E.coli activates ion transporter
channel and start transport of salt ion outside from bacterial cell. The
activation of this transpoter channel occur only when it gives induction with a
comple of KdpD and KdpE, when phosphorylation occured this complex is formed (Gelvin, 2003; Hood et al., 1986). The universal stress protein also having ATP binding motifs which is
present on its N terminal. But th C terminal region takes different form
depends upon the protein which intract. But in same cases C terminal also binds
with ATP molecule due to this binding
USP capables to bind with 2 ATP molecule. Some Universal stress proteins have
good percentage of glycine aminoacid
which allows universal stress protein intract with other proteins (Li et al., 2005). Our research was based on universal stress protein 2 (USP2) which helps
plant under stress condition to maintain itself, universal stress protein2
provids resistance against drought, heat, osmotic and salt stress. Universal
stress protein2 was islolated from Gossypium
arboreum and transformed in E.coli
for its expressional studies.
Material and methods
Place of study/research
All the reseach work of cloning was done in moleculer lab of CRiMM department of
the Unversity of Lahore. Luria bertan (L.B) media was prepared by using diffent
ingridents like Yeast
extract 5g/l, NaCl 10g/l, Tryptone
10g/l and Agar used to solidify the media was 0.5% and autoclaved it on 1210C
temperature, 15 Pascal pressure for 20 minute to avoid any type of contamination,
after autoclaving supplemented with
different antibiotics like kanamycin 50mg/ml and ampicillin 100mg/ml for bacterial growth. This media was
used to grow bacteria contained all the nutrients required for bacterial
growth.
Sterilization of
working area
The surface of laminar flow cabinet was
sterilized by using 70% ETOH use as a disinfectant after media preparation and
autoclaving of the media placed in laminar flow cabinet and other things which
was used during inoculation of bacterial culture process like Pipette,
spreader, colony picker, petri plates, paraffin,
scissor, pipette tips, and others. After placing the material and disinfection
of the cabinet turn on UV light for 20 minute to kill microbes present on the
surface of things.
Revival of stock
of bacterial culture
Liquid broth
After
media preparation inoculated the bacterial culture for its maximum growth and
for plasmid isolation, PCR reaction etc. take 10 ml of freshly prepared L.B
media in 250ml flask, add 1ml of stock culture of each (puc,
pet, top10, bl21) culture, supplemented with 10 µl antibiotic which is specific
according to bacterial strain and was placed on shaker incubator overnight at
370C and 250 rpm.
Solidifying
media
After autoclaving of media, it was placed
in laminar flow cabinet to cool down and to avoid contamination. When media is
bear able to touch add required antibody and pour it in plastic petri plate, uncovered the petri
plate until media is solidify. Take 5µl of stock culture and spread it on petri plate with the help of spreader, after this wrap petri plates with paraffin and placed petri
plates in incubator temperature was 37oC overnight for bacterial
growth.
Strains of
bacteria and plasmids
Different
strains of bacteria and plasmids are used which are listed below.
Isolation of
plasmid: After
revival of culture the next step was isolation of plasmid by using plasmid
extraction kit method.
Gel
electrophoresis
Gel
electrophoresis is another technique which was used to identify isolated
plasmid quantity and it is also used for PCR product conformation, run with 1Kb
ladder to conform the size of the product. First make 50X TAE (tris acetate EDTA) stock concentration then diluted with
distilled water and convert 50X TAE into 1X TAE (working concentration). Take
20ml of stock of 50X TAE and dissolved it into 80ml of distilled water to make
1X TAE. Dissolved above given
quantity of agarose (used as solidify agents) into
required ml of TAE and heat till agarose was
dissolved properly after this wait till the gel was bear able to touch and add ethedium bromide and
mix them well and pour into gel caster where the comb was already set before
pouring gel. When gel was solidify remove comb and pour sample into gel well
with dye (bromo phenol blue) and also run 1Kb ladder,
after loading run gel at 100 voltage for 30 min from negative pole to positive
pole to identify required segment gel was observed under UV transilluminator.
PCR (polymerase
chain reaction)
Polymerase
chain reaction was the next step after plasmid isolation and conformation of
plasmid by using gene specific primer to amplify specific segment at specific
temperatures.
Restriction of
plasmid and gene
After
conformation of plasmid and amplification of specific gene segment the next
step was restriction, restrict plasmid and gene with two different enzymes to
avoid frame shifting and for normal functioning protein. The restriction is
done with enzyme Nde1 and BamH1 which produced sticky ends, or overhangs have
same complementary sequences. After preparing
restriction mixture incubate PCR tubes at 37oC
for 2 hours after this perform gel electrophoresis to observed restricted
fragments on gel.
PCR purification
method
After
restriction of plasmid and gene cut the segment of plasmid and gene present on
gel with the help of cutter under UV transluminator
and put in eppendroff, after cutting purify the
nucleic acids by using quegene kit method, in this
kit method different buffers are used to purify contents.
Ligation
After
nucleic acid purification the next step is ligation of the plasmid with gene
with the help of enzyme T4 DNA ligase. Ligation was
done after preforming nanodrop
(which is used to detect nucleic acid concentration in elution buffer). The nanodrop value for PUC (which contain USP2 gene) is 6.1ng/
µl, and nanodrop value of pet vector is 12ng/ µl. the
ligation is done with 1:2. After
preparing reaction mixture incubated overnight at 22oC, after
overnight incubation speared whole reaction sample on kanamycin
contain L.B agar plate and incubated overnight
Competent cells
formation
After
ligation next step to prepared competent cells of bacterial strains top 10, for
preparing competent cells or the transformed cells was speared on kanamycin contain L.B agar plate and incubated overnight, after
incubation first growing colony pick and cultured into L.B media, isolate
plasmid and perform PCR to conform USP2 gene ligation and transformation.
Results and
discussions
Isolation of
plasmids (PUC57 and PET 30b)
This
gel electrophoresis shows the isolated plasmid from stock solutions of
bacteria. Plasmids were isolated by using plasmid extraction kit method and
stored at -20. The size of Puc plasmid was 3220b.p
and size of Pet 30b was 5400bp (Figure 1).
Conformation of
universal stress protein through PCR
After
isolation of plasmids the PUC plasmid was amplified through PCR by using gene
specific primer and run along with 1Kb ladder to confirm the size of gene which
was about 500 base pair long (Figure 2).
Restriction
of plasmid and gene
After
conformation of the gene in puc plasmid the next step
was restriction of Pet plasmid and universal stress protein with Ndel and BamHl which produce
sticky ends to avoid frame shifting (Figure 3).
Ligation and
transformation
After
restriction the next step was ligation of plasmid with gene with the help of T4
DNA ligase at 22oC overnight. Transform ligated cells into freshly prepared competent cells, and
spread it on ampicillin containing nutrient agar
plate (Figure 4).
Conformation of
the clone of universal stress protein through PCR
After
transformation colonies grown on ampicillin
containing agar plate were recultured into broth of
nutrient media at 37 oC
overnight. Then plasmid was isolated and amplified through PCR to confirm clone
of gene (Figure 4 and 5).
The
results from figure 6, revealed that there was a
gradual increase in the expression of USP2 gene for heat stress in E. coli with passage of time interval of
2 hours to 12 hours. The non-transgenic cells were died in the culture while
increasing the duration of heat stress. It was also found from figure 7 that
there was also gradual increase in the osmotic adjustment in the transgenic
bacterial cells as compared with non-transgenic bacterial cells. Similarly, the
tolerance of transgenic bacterial cells was enhanced with increasing time
interval as compared with the non-transgenic bacterial cells.
Figure 6. Transgenic and
control/no-trangsgenic E.coil for
heat stress
Figure 7. Transgenic and
control/no-trangsgenic E.coil for
osmotic stress
Figure 8. Transgenic and
control/no-trangsgenic E.coil for
salt stress
Discussions
The
transformation technique is used to protect crop plants from abiotic and biotc stress
conditions which help in minimizing the damage through stress conditions in
crop plants (Howe and
Jander, 2008; Lawrence and
Novak, 2006). It Provides defense mechanism
against stress. Transformation of USP2 gene was done with the tag of ubiquitin promoter (Jabeen et
al., 2008; Sultan et
al., 2009) along with report gene like GUS
gene. In our study the report universal stress protein was successfully
transformed into vector expression strain of bacteria by using pet vector which
was restricted with Nde1 and BamH1. The restriction which is produced sticky
ends and ligation with T4DNA ligase. The ligated universal stress protein gene is very close to
vector promoter region to obtained maximum expression and number of copies.
Universal stress protein helps to provide resistance in plant against drought
tolerance. Similar results have been reported in various studied (Ibl and
Stoger, 2012; Reyes et al.,
2010). To achieve the
maximum transformation rate efficiency was carried out by changing the media
components concentration by providing kanamycin
250mg/l (Grover, 2012;
Li and Greene, 2010). But in this research, the
maximum growth of bacteria is obtained by using optimized concentration of
bacterial culture media with 50mg/ml kanamycin. It has been found that cloning of gene is usually a
major step to transform gene in crop plants with desireable charecteristices.
Universal stress protein helps to maintain water level in plant cells specially
in cotton to provide drought resistance. Universal stress protein is a
regulatory protein and its activity is increased by introducing its intractions
with other morphological, moleculer and
biochemical traits (Jin et al., 2010; Yang et al., 2014). The transformed bacterial cells which showed tolerance under heat
stress, osmotic stress and salt stress conditions indicated that the cells with
USP2 gene shows tolerance against changing environmental conditions. The
abiotic stress under such conditions of transgenic bacterial cells may indcue
the change and ability to withstand against stressful environmental conditions (Engel et al., 2014; Pouly et al., 2013; Rossier et al., 2013). The use of USP2 gene for crop
plants may be helpful to produce abiotic stress tolerance in crop plants to
increase crop plant yield. It was suggested from our study that the USP2 gene
may be used to produce transgenic crop plants against abiotic stress
conditions.
Conflict of intrest
The
authors showed absence of conflict of interest for manuscript publication.
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