Plant Genetic Engineering

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Plant Genetic Engineering
Sun Yue
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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.
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• 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.

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General procedure
Gene cloning
Vector Construct
Plant transformation (Regeneration)
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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.
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Want more? You may be interested in

• Genome walking
• RACE (Rapid Amplification of cDNA ends)

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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. 
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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 semi-random location into the
plant genome.
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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. 

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Vector Construct Binary vector system
binary plasmid (Expression/shutle vector)
T-DNA binary system 
helper plasmid (Disarmed vector)
Engineered A. tumefaciens strains (EHA105/LB4404
)
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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.

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GMO verification
Molecularly
Physiologically and Biochemically

• PCR
• Southern blotting
• Real-time PCR
• Western blotting

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Some part of my work
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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

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Trichomes

• Trichomes can be superficially regarded as
  Plants 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.

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NtLTP1
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Partial results in this paper
Fig 1.
Fig1. (c,d,e) GUS expression in leaves of
transgenic tobacco plants with NtLTP1proGUS (g)
Leaf surface and (h) Root of transgenictobacco
plants expressing NtLTP1 with 35SproGUS
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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.
• Microscopic view of trichome exudates in
  NtLTP1-overexpressing plants.
• Trichome exudates from NtLTP1-RNAi plants
• Trichome exudates from wild-type plants
• Volumes of trichome exudates in
  NtLTP-overpressing or RNAi transgenic lines.

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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.

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The features of NtLTP1
Fig 5.
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.

• Levels of NtLTP1 transcripts were approximately
  3.1-fold higher in trichomes than in leaves
  without trichomes. Its glandular trichome
  specific.

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A shortcut
All I want to do is trying to endow soybean with
characteristic of aphid-resistance. Here, since
NtLTP1 in this article has been confirmed to be
lipid-transferred and resistant to aphid, a quite
straightforward way is inserting the
NtLTP1-overexpression construct directly into
soybean. Its 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.
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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
  havent 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.

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Protein sequence comparison
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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/ ).

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Fig 9. Conserved domains and Functional motifs
analysis indicates No.4,6,7 are the most
identical to NtLTP1
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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)
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Advanced protein structure comparison (No.6
NtLTP 1)
GmLTP-like6
Fig 11. Protein tertiary structure comparison
between No.6 and NtLTP1 from different
perspectives. (Expasy Prediction)
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• 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.
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• 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

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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 hasnt yet been discovered. According
  to the result of field experiment conducted by
  Hong (Hong et al., 2010), the trichome type
  doesnt 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 its reasonable to believe that
  lipid and related metabolites are greatly
  involved during aphid-resistance process.

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Some results

• The GmLtp-like cloned from soybean
• PCR verification about 35SGmltp-like construct
• Enzyme digestion of 35SGmltp-like construct
• Germ-free tobacco seedling
• Differentiation from infected tobacco leaf
• Regeneration bud of infected tobacco