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Plant Genetic Engineering
Sun Yue
Plant Genetic Engineering
Genetically modified crops (GMCs, GM crops, or biotech crops) are
plants, the DNA of which has been modified using genetic
engineering techniques, to resist pests and agents causing harm to
plants and to improve the growth of these plants to assist in farmers
efficiency.
• Both conventional and GM plant breeding
change the genes of a plant so that a new and
better variety is developed.
• GM plant breeding changes the genes of a
plant more precisely and efficiently than
conventional plant breeding.
General procedure
Gene cloning
Vector Construct
Plant transformation
(Regeneration)
Gene cloning
PCR (Polymerase Chain Reaction)
The polymerase chain reaction (PCR) is a biochemical technology in molecular
biology to amplify a single or a few copies of a piece of DNA across several orders
of magnitude, generating thousands to millions of copies of a particular DNA
sequence.
Want more? You may be interested
in …
• Genome walking
• RACE (Rapid Amplification of cDNA ends)
Vector Construct – Plasmid & Vector
Plasmid - small circular, double-stranded DNA molecules in bacteria, In nature,
plasmids carry genes that may benefit survival of the organism.
Vector - Plasmids used in genetic engineering are called vectors. They are commonly
used to multiply (make many copies of) or express particular genes.
Vector Construct – Agrobacterium tumefaciens
Agrobacterium tumefaciens is the causal agent of crown gall disease (the formation
of tumours) in over 140 species of dicot. It is a rod shaped, Gram negative soil bacterium.
Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA,
for 'transfer DNA'), from a plasmid, into the plant cell, which is incorporated at a semirandom location into the plant genome.
Vector Construct – Binary vector system
• In order to be virulent, the bacterium
must contain a tumour-inducing
plasmid (Ti plasmid or pTi), of 200 kb,
which contains the T-DNA and all
the genes necessary to transfer it to
the plant cell.
• The DNA transmission capabilities
of Agrobacterium have been vastly
explored in biotechnology as a means
of inserting foreign genes into
plants.
Vector Construct – Binary vector system
binary plasmid (Expression/shutle
vector)
T-DNA binary system
helper plasmid (Disarmed vector)
Engineered
A. tumefaciens
strains
(EHA105/LB44
04)
Plant regeneration – plant tissue culture
•
Plant tissue culture is a collection of techniques used to maintain or grow plant
cells, tissues or organs under sterile conditions on a nutrient culture medium of
known composition.
•
Based on the development of the Plant Cell Totipotency.
GMO verification
Molecularly;
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•
•
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PCR
Southern blotting
Real-time PCR
Western blotting
Physiologically
and
Biochemically:
Some part of my work
Aphids
• Aphids are small sap sucking insects, and members of the superfamily
Aphidoidea. They are tiny and fragile but make it to survive under
treacherous nature due to the feature of asexual reproduction.
• The soybean aphid (Aphis glycines) is native to Asia.It is described as a
common pest of soybeans in China.
• Heavy infestations of soybean aphids may cause problem such as plant
stunting, premature defoliation, lower seed weight etc.. Yield losses as
high as 50 to 70% have been documented as a result of prolonged
exposure to high densities of soybean aphids
Trichomes
• Trichomes can be superficially regarded as “Plant’s hair” and fall into two
categories which are Glandular trichome and Non-glandular trichome.
• Glandular trichomes affect the plant in a number of ways. It contains or
secretes a mixture of chemicals that can be used as pesticide, pharmaceutical
and flavour/fragrance industries. Besides glandular trichomes on some crop
species confer resistance against insect pests.
• One of the exudates from glandular trichomes is lipid.
NtLTP1
Partial results in this paper
Fig 1.
Fig 2.
Fig1. (c,d,e) GUS expression in leaves of transgenic tobacco plants
with NtLTP1pro::GUS
(g) Leaf surface and (h) Root of transgenictobacco plants expressing
NtLTP1 with 35Spro::GUS
Fig2. Localization of the NtLTP-GFP fusion
protein
To examine the biological function of NtLTP1 in glandular trichome secretions,
transgenic tobacco overxpressing NtLTP1 and NtLTP1 RNAi were constructed.
Fig 3.
Fig3. Liquid droplets secreted
from trichomes in transgenic
and wild-type tobacco plants.
(a) Microscopic view of
trichome exudates in
NtLTP1-overexpressing
plants.
(b) Trichome exudates from
NtLTP1-RNAi plants
(c) Trichome exudates from
wild-type plants
(d) Volumes of trichome
exudates in NtLTPoverpressing or RNAi
transgenic lines.
Aphid choice tests
Fig 4.
• Transgenic tobacco plants
overexpressing NtLTP1 demonstrate
enhanced aphid resistance.
• The NtLTP1 gene may play an
important role in active lipid
secretion from long glandular
trichomes in tobacco.
• Genetic manipulation of NtLTP1 may
result in genetic engineering
techniques that could stimulate the
production of useful products from
glandular trichomes in economically
important crops.
The features of NtLTP1
Fig 5.
 Levels of NtLTP1 transcripts were
approximately 3.1-fold higher in
trichomes than in leaves without
trichomes. It’s glandular trichome
specific.
Fig 6.
 Purified recombinant NtLTP1 binds the
TNS lipophilic probe. Black squares
indicate recombinant NtLTP1 proteins,
white squares indicate the protein buffer
control. Results suggest that NtLTP1 is
able to bind lipids.
A shortcut
All I want to do is trying to endow soybean with characteristic of aphidresistance. Here, since NtLTP1 in this article has been confirmed to be lipidtransferred and resistant to aphid, a quite straightforward way is inserting the
NtLTP1-overexpression construct directly into soybean.
It’s probably the safest way to achieve the goal.
But by doing this, an article of fine IF is not likely to be obtained. After all I
“borrowed” the fruitful achievement from others. Also I need to take the
time-consuming soybean transformation into account.
But…
There is LTP gene in Nicotiana tabacum, then, is there LTP gene in
Glycine Max? I searched for it in NCBI database, finally I found
there is a total of 7 candidate genes of which function haven’t been
proved yet. They are predicted as “GmLTP-like”, but fall into the
group of Non-specific lipid-transfer protein 3-like. I named them as
No.1-No.7 and compared the amino acid sequence with each other.
It turns out the identity is pretty high.
Protein sequence comparison
Fig 7.
Alignment of NTP-like (No.1-7) protein sequences from Glycine Max
Fig 8.
Alignment of NTP-like protein sequence (exclude No.5) with NtLTP1
In order to find out which is the most identical to NtLTP1,
the next processes were carried on including:
 Analysis for conserved domains and functional motifs by using
Inter ProScan ( http://www.ebi.ac.uk/Tools/pfa/iprscan/ ).
 Physicochemical properties analysis by using DNAMAN software.
 Advanced protein structure prediction by using Expasy
( http://swissmodel.expasy.org/ ).
 Gene expression analysis in SoyBase database
(http://soybase.org/soyseq/ ).
No.1
No.2
No.3
No.4
No.6
No.7
Fig 9. Conserved domains and
Functional motifs analysis
indicates No.4,6,7 are the most
identical to NtLTP1
NtLTP1
No.6 was selected and performed related protein properties
analysis
NtLTP1
No.6 GmLTP-like
Fig 10. Protein
properties
analysis. ( Amino
acid composition,
Protein
secondary
structure and
Protein
hydrophobic &
hydrophilic
analysis)
Advanced protein structure comparison (No.6 & NtLTP 1)
GmLTP-like#6
NtLTP1
Fig 11. Protein tertiary
structure comparison
between No.6 and NtLTP1
from different perspectives.
(Expasy Prediction)
Gene expression of No.6
Fig 12. Searching expression status of No.6 from SoyBase (http://soybase.org/soyseq/).
The result illustrates in seeds of soybean, from 10th day onwards, expression of the interest
gene grows dramatically and reaches the highest level when the time has come to 35 days,
which is consistent with the predicted function of Lipid Transfer Protein.
 By predicting related information of GmLTP-like #6, seemingly I found the gene in
soybean which is the most similar to NtLTP1 in terms of amino acid sequence and
function. Also according to the result of gene expression status from SoyBase , the
possibility of pseudogene is ruled out. However, further validation of gene function is
necessary. For now general strategies designing to achieve this purpose are as
followings:
Gene function validation
P35S
Physiological and
biochemical analysis
GmLTP-like
Arabidopsis transformation
NtLTPpro
Promoter function
analysis
GUS
NtLTPpro
GmLTP-like
Soybean transformation
Aphid-resistance
authenticity experiment
Conclusion
• Trichomes being as the first defense system facilitate plants against from pests either by
means of morphological obstacles or related metabolites. But the particular mechanism
of pest resistance hasn’t yet been discovered. According to the result of field
experiment conducted by Hong (Hong et al., 2010), the trichome type doesn’t affect
soybean aphid (Aphis glycines) reproduction a lot. It may imply the morphology of
trichome is not the major concern for aphid-resistance.
• Correlation studies indicate that triterpene possesses insecticidal activity towards
aphids (Ellen et al., 2011). Wax on plant surface can also provide defense against pests
and herbivores by reducing their ability to attach to plants (Eigenbrode, 2004). Both of
these two substances (triterpene, wax) belong to lipid family. Moreover Joe identified
two genes that regulate metabolism of lipid synthesis in Arabidopsis thaliana, and
conferred resistance to aphid (Joe et al., 2010).
• In conclusion it’s reasonable to believe that lipid and related metabolites are greatly
involved during aphid-resistance process.
Some results
a)
The GmLtp-like cloned from
soybean
b)
PCR verification about
35S::Gmltp-like construct
c)
Enzyme digestion of
35S::Gmltp-like construct
d)
Germ-free tobacco seedling
e)
Differentiation from infected
tobacco leaf
f)
Regeneration bud of
infected tobacco