UNIVERSITY these stresses is complicated and changes involves at

    Centre of
Agricultural Biochemistry and Biotechnology

for M.phil Biotechnology)

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    Title: Molecular Characterization of NHX gene in sugarcane  

     Name of student             :                   Muzammal zohaib

     Registration No              :                   2011-ag-4033

     Name of Supervisor       :                   Dr. Ghulam Mustafa





of Agriculture, Faisalabad
of Agricultural Biochemistry and Biotechnology

(Synopsis for M.Phil

Characterization of
gene in sugarcane 


                         Date of Admission:                 16th September 2016

                         Date of Initiation:                    16th September 2016

                         Probable Duration:                   4 Semesters


a)     Name
of student:       Muzammal Zohaib

b)    Regd.
No.           :       2011-ag-4033

  Supervisory Committee:

1.         _________________________      Chairman


2.         ­­­­­­­­­­­­­­­Dr. Ghulam Mustafa                       (supervisor)


3.         Dr. Faiz Ahmad Joiya                     (member)


4.         Dr. Muhammad afzal                      (member)


for the Project


is the member of grass family, Poaceae,
belonging to the genus Saccharum,
classified under the Andropogoneae tribe; with maize and sorghum, as the
closest relatives (Plomion et al., 2011). The
plant of sugarcane is the most important source for the production of about 80%
sugar needed by the world. This study objective is to extraction and
identification of DNA from sugarcane. And characterize the NHX gene and also
find out the effect of Na+/K+, Na+/H+
antiporters after encoding by the NHX gene under saline conditions.
Furthermore, different bioinformatics tools will be used to characterize the
NHX gene.

70% crop productivity can be decreased every by abiotic stresses (Acquaah 2007).
Plants response towards these stresses is complicated and changes involves at
molecular levels by using different molecular techniques at proteomics,
transcriptomics, at physiological and cellular levels. It is synchronized
action of genes against stress tolerance, one of the gene can triggered
components of different pathways (tuteja, 2007). Salinity stress is one the
basic and major stress among all the abiotic stresses. Growth and production of
many crops can be affected by salinity stress in over the world (Geo et al., 2007). More than 800 million
hectares of land was affected worldly by salinity stress (FAO 2008, Different mineral nutrients required for
the growth of plants, but Na+  isn’t considered as essential mineral
nutrient, moreover, excess of Na+ adversely affect the growth of
plants and can cause the lost of yield and also delay the flowering of plant
(Gill 1979, Hasegawa et al., 2000,
Zhu 2001, chinnusamy et al., 2006). Mostly
salin soils are rich in Na+ which distrupts the absorption of K+
and other minerals (Zhu 2001). Brazil is at first number in the production
of sugarcane with the yearly production of 739,300 TMT.

Like other crops sugarcane is also a major crop and it
is also a source of income for most of peoples about 9 million Pakistanis. Pakistan’s
government has already taken many steps with the passage of time to improve the
country’s general production. These steps have improved the overall sugarcane
production therefore our country is growing up to of 63 to 64 thousand metric
tons of the goods. This step made the Pakistan a well reputed country and gave
the 5th position in world as sugarcane producer. So, now Pakistan is
exporting sugar to the next-door countries as Afghanistan, Tajikistan, and other Asian countries.

A-biotic stress:

Sugarcane (Saccharum species hybrids) is a lengthy or time
taking and required excess of water for this cash crop to cultivate under
varied agro-ecological environment. Moreover experiencing different weather
environment all the year round, alteration in climate, the range of the day,
further ecological condition cause the development of sugarcane by distressing
the abiotic stresses, growth of sugarcane, sugar production, its growth and
revitalization, ratooning capability and accessibility of sugarcane seed for
successive planting. The comparatively more litheness of sugarcane towards
abiotic stresses appears due to some native changing as a good array of
compensatory potential, C4 photosynthesis, require privileged
temperature for the majority of the physiological behavior (but for sugar
increase). Besides, a number of the genetic or physiological interventions as
providing the drought hardiness, helping of roots to penetrate deeply in soil,
heat load can be condensed by litter mulching, increasing the age of the crop
at the beginning of dearth/floods, by amendmenet of organic matter in soil,
improvement in nutrients and managing the rhizospheric
salinity/alkalinity, etc., also improve to stress tolerance in sugarcane. Moreover,
there are number of genes, miRNAs
techniques and different molecular markers are associated to control these stress
responses which contribute to buoyancy of sugarcane towards abiotic stresses.
Such efforts will lead to development of a stress resistance variety of
sugarcane utilizing NHX gene, imparting salt tolerance
esapcially Na+/K+, for commercial cultivation.

 Seed performance can
be improved by seed priming a well known pre-germination strategy. However,
biochemical and molecular mechanisms underlying priming mediated stress
tolerance are little understood. Here, I’ll report results of the study on
growth, physiological characteristics, the growth pattern and expression of
stress reactive genes in salt primed. The antioxidant capacity, osmolytic
improvement and unpredictable growth were exposed between the primed and
non-primed plants. The prepared plants showed better forbearance towards the
salt or PEG stress, as showed by better development and lower membrane breakage,
through better antioxidant capacity as compared to the respective non-primed
controls. Further, steady state transcript expression analysis revealed up regulation
of sodium-proton-antiporter (NHX),
while down-regulation of sucrosetransporter (SUT1), delta1-pyrolline-5-carboxylate-synthetase (P5CS) and proline-dehydrogenase (PDH) in primed plants on exposure to the
stress as compared to the non-primed plants. By altering gene expression the
results showed that primed mediated salts and PEG stress tolerance have improved
the antioxidant capacity in sugarcane.

NHX Gene:

                 The  biochemical and
physiological information recommended that Na+/H+ and K+/H+ antiporters are concerned to regulate
the pH by fluctuating the intracellular ions intracellular ion, it was a sturdy
and time taking process to find out these genes which encoding antiporters.
Through advancement in gene understanding from 2000 when Genome sequencing
projects launch revealed that there are number of putative Cation/Proton
antiporters in plants, their function is to be still studied for better
improvement in plants. These intracellular NHX transporters which constitute
the first one Cation/Proton exchanger family was studied in plants. The
beginning member which was to be found, AtNHX1, was recognized as a significant
salt tolerance determinant and recommended to catalyze the Na+ accumulation in vacuoles. Now it is,
though, fetching clear, that this gene and some other members of the same
family also play vital roles to regulate pH and K+ homeostasis, regulation methods from
vesicle trafficking and cell development to plant growth. Tough
Na+/H+ antiporter promote the simply di- or
tripeptide transporter function. The transfer of
a solute or solvents from outer to inner side of a membrane or to the other
happened according to given reaction  K+(in) + H+(out) =
K+(out) + H+(in).


To develop a variety of
sugarcane against abiotic stress especially to control Na+/k+ ions
in sugarcane under saline soil.

Materials and methods:

planned research study will be carried out in Transgenic Laboratory at Centre
of Agricultural Biochemistry and Biotechnology (CABB), University of
Agriculture, Faisalabad under the supervision of the most honorable supervisor
Dr. Ghulam Musatafa.

Plant material:     Plant material which will be used in the study is provided by
my supervisor from a special variety of sugarcane grown under controlled

DNA isolation from sugarcane leaves:

Reagents needed:   1). CTAB buffer           2).       2% CTAB 20gm CTAB

20mM EDTA 40ml EDTA stock (0.5M)    
4).       100mM Tris-Cl pH 8.0
100ml Tris-Cl stock (1M)

1.4M NaCl 280ml NaCl stock (5M)             6).        + 0.2% Mercaptoethanol

Make up to 1 Litre with water, pH 7.5 – 8.0, and autoclave     8).  
76% Ethanol

DNA extraction:

First of all I’ll take
20-25g leaf sample then wash it d3H2O

I’ll grind the sample
by pestle and morter by adding CTAB+ B-merceptoethanole

Incubate that sample
at 65Co for 30 minutes in water bath

Then it will be
centrifuged at 13200rpm for 3 minutes

Then take supernatant

Add equal volume of

Again centrifuged it
13200rpm for 3 m

Repeat this step one

Then add 2/3 vol. of

Keep it at -20 or -40
Co for 30 m

Centrifuges it at full
speed for 15 m

Discard supernatant
and take the pallet

Add 200-300ul of d3H2O
or R-40

In case of d3H2O
it will be treated with RNAs at 37Co for 1-3 hours

Now it will be stored
at -4 or -20


It is used to
amply the DNA. After extraction PCR will
performed by different methods e.g. temperature gradient or simple one to get
the desired gene or required DNA fragments.

The NCBI database will be used as a
source for nucleotide sequencing and protein analysis. TMpred will be used for
the prediction of transmembrane domains and Clustwal W for sequence alignment. PSIPRED
protein structure prediction tool will be used for the prediction of secondary
structure. Expasy ScanProsite ( will be used for the prediction of Phosphorylation
motifs. MEGA 3.1 will be used for phylogenetic analysis. PCR will be used to
amplify DNA fragments.

Molecular analysis:

A specific variety of
sugarcane was already sown in pots under controlled conditions.

DNA will be extracted
from the fresh leaf samples by following the method reported by Khan et al. (2004).

Quantification of DNA
will be optimized.













































             Name of student:      Muzammal zohaib                      ____________

                  Supervisory committee (name
& signatures)

1.     Dr.
Ghulam Mustafa             (Supervisor)         ____________


2.     Dr.
Faiz Ahmad Joiya         (Member)               ____________


3.     Dr.
Muhammad Afzal        (Member)               ____________ 


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