Question stem cell culture or protoplast culture instead

Question or problem to be addressed 50

Background to the problem 300

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Experiment to address the problem 300

Expected outcomes and alternative strategy if necessary 100



Researchers generate hybrid crops for enhanced production
which are not stress tolerant as compared to wild breeds. However,
transcription factors like NAC (NAM, ATAF1,2 and CUC2) regulate the expression
of the stress responsive genes. Could it be feasible to manipulate the genetic
architecture using these transcription factors against various stresses?


proteins act as transcription factor and regulator of stress tolerance genes.
Researchers identified NAC proteins from Arabidopsis, rice (Oryza
sativa), soybean (Glycine
max), wheat (Triticum
species),poplar (Populus
trichocarpa) and citrus (Citrus sp.).
A very small number of NAC proteins also found in
conifers, pteridophtyes (Selaginella
moellendorffii) and moss (Physcomitrella
patens). NAC proteins have a
N-terminal NAC domain and a C-terminal domain, which regulate the
transcription. Sometimes, NAC domain contain a negative control region and the
C-terminal contain a transmembrane motif described by Swati Puranik et al.
(2012). Despite of a common structure and motif, these proteins are plant
specific, and in various plants, NAC dependent stress tolerance pathway may
vary. In rice, OsNAC5 transcription factor controls the stress tolerance by
regulating stress-inducible genes. OsNAC1 – OsNAC8 genes (total 8 genes)
described by Kikuchi et al. (2000). Ooka et al. (2003) described 75 predicted
NAC proteins in rice and 105 predicted protein in Arabidopsis. All NAC coding
gene identification, sequencing and its interaction with stress tolerance genes
will be helpful to figure out the total stress tolerance pathway of plants.
Except the model plants, other plants may show variation from the pathway, and
that also can be another study.



1. Expression pattern from NGS study and checking in BLAST

2. SiRNA to validate in vitro and in vivo experiments

3. Protein sequence and structure  prediction from iTRAQ, comparative

4. Function from genetic manipulation under native and 35S

5. Study of pathway using gene pyramid transgenics


As a small portion of total genome is studied,
identification of the all NAC coding gene, mutant screening or insertion of a
transgene methods will be very tedious. It can be a new approach to use the si
RNA for gene silencing assay and use of plant leaf and stem cell culture or
protoplast culture instead of growing a whole plant batch again and again.
Plant cell suspension culture will not be helpful, because those cells remain
in undifferentiated stages. Exogenous si RNA tagged with Green Fluorescent
Protein can be applied to the cell culture or protoplast culture of plant leaf
and stem cells. Then the cells can be placed in various increasing or decreasing
grade of stress (example: Salinity stress). Already sequenced and predicted NAC
family gene silencing by si RNA along with the total proteome study,
isoelectric focusing and 2D gel study of control plant cell culture or
protoplast culture; and the comparison of total proteome of control and treated
culture will show the difference in stress tolerance.



NGS study of different plants and matching those sequences
with the already sequenced NAC protein coding genes will show the differences
between the sequences and the primary idea about the variation of NAC coding
gene from plant to plant. From the sequence, the probable protein sequence can
also be done. From these predicted data, protein pI and 3D modelling of protein
can be done by a computational approach (such as: Homology modelling), which
can help us in the total proteome study result prediction. If there is a huge
difference, we can go for X-ray crystallography.









To adding the stress tolerance property to the hybrids, we
must know the whole NAC protein structure, NAC coding gene position,
activation, transcription and deactivation of the genes of wild crops.