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.

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

Plasmid - small circular, double-stranded DNA molecules in bacteria, In nature, plasmids carry genes that may benefit survival of the organism.

Vector Construct – Plasmid & Vector

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 semi-random 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

T-DNA binary system

binary plasmid (Expression/shutle vector)

helper plasmid (Disarmed vector)

Engineered A. tumefaciens strains(EHA105/LB4404)

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

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

Molecularly; Physiologically and Biochemically:

Some part of my work

• 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

Aphids

• 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.

Trichomes

NtLTP1

Partial results in this paper

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

Fig 1. Fig 2.

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 NtLTP-overpressing or RNAi transgenic lines.

• 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.

Fig 4.Aphid choice tests

The features of NtLTP1

Levels of NtLTP1 transcripts were approximately 3.1-fold higher in trichomes than in leaves without trichomes. It’s glandular trichome specific.

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.

Fig 5. Fig 6.

A shortcutAll 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. 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.

Alignment of NTP-like (No.1-7) protein sequences from Glycine Max

Alignment of NTP-like protein sequence (exclude No.5) with NtLTP1

Fig 7.

Fig 8.

Protein sequence comparison

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

NtLTP1Fig 9. Conserved domains and Functional motifs analysis indicates No.4,6,7 are the most identical to NtLTP1

No.6 was selected and performed related protein properties analysis

Fig 10. Protein properties analysis. ( Amino acid composition, Protein secondary structure and Protein hydrophobic & hydrophilic analysis)

NtLTP1 No.6 GmLTP-like

Advanced protein structure comparison (No.6 & NtLTP 1)

GmLTP-like#6 NtLTP1Fig 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 GmLTP-like

NtLTPpro GUS

NtLTPpro GmLTP-like

Promoter function analysis

Soybean transformation

Arabidopsis transformation

Physiological and biochemical analysis

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