Background

China is one of the biggest agricultural countries over the world. But in our country, farmers still have a lot of problems to deal with. Drought, pests, water pollution, weeds and so on. These problems cause damage to the agriculture industry. So we want to do some help.

We focused on the weeds problem. Usually, farmers use herbicides to kill the weeds. However, the abuse of herbicides in our country have caused a series of secondary prblems. One of them is that the weeds have the resistant to the herbicides. As we all know, if we always use one kind of antibiotic,the bacteria will become drug-fast. So do the weeds. If famers use only use one kind of herbicide, as the time goes by, the weeds will become herbicide resistant. And farmers have to raise the dose to kill them. However, the more dose farmers raise, the stronger the weeds will be. It is a vicious circle. What’s more, raising the dose will cause many other environmental problems. It will raise the poisonous residue and do harm to animals and human.

Let’s use the antibiotic as the example again. If we find that the bacteria has the resistant to antibiotic A, then we will antibiotic B to kill it because there is very few chance that the bacteria have double resistance at the same time in the wild. Similarly, if we use two or more kinds of herbicides the resistant weeds will be killed easily. Also the mix use of herbicides can help us solve many other probelems. So, obviously, Creating a genetically modified crop which is resistant to more than one kind of herbicide enable us to utilize a combination of several herbicides in agriculture.So, obviously, Creating a genetically modified crop which is resistant to more than one kind of herbicide enable us to utilize a combination of several herbicides in agriculture.

How did we come up with it?

Unlike the microrganism, co-expression of diffrent kinds of genes is hard to achieve for plant(without a counterpart of operon ) . In our project we choose 2A to serve as a linker between 2 proteins. 2A is a short peptide containing 20 contiguous amino acids. And it has an interesting feature.When placed between 2 genes using a single ORF, during the translation process, the attachment between 19th and 20th amino acid will break. Then the translation process will continue. That means 2A provides us a potential approach to realize gene co-expression.

The major advantages of using the 2A system in the construction of multicistronic vectors are(i)its small size(54-174bp)compared to IRESes,that coexpression of proteins linked by 2A is independent of the cell type(since structurally highly conserved amongst the eukaryotic ribosomes,structurally highly conserved amongst the eukaryota),and(iii)that multiple 2As may be used, the activity of each being completely independent.

Our project create 4 genetically modified Arabidopsis thaliana, and categorized them into 2 combinations, one being BAG & GAB, the other BGT & TAB(B=BAR G=GAT T=tfdA, they're 3 common herbicide resistant genes. A=2A linker). We hope 2A will help with the puzzle of herbicide resistance in agriculture.

Pre analysis

Before we started to do our in-lab experiments, we did some analysis of 2A system to help us build a better device. According to previous reports and our analysis, these factors will influence the efficiency of 2A cleavage: (i) 2A sequence (ii) upstream sequences of 2A (iii) The length of 2A.

As for the sequence of 2A, we analyze the sequence of several natural different 2A linkers. The result shows below.

So there are several conserved residues in 2A sequence, site mutant experiment carried by Ryan shows that mutant of these conserved residues may lead to much lower cleavage efficiency.

In order to know why upstream sequence and length of 2A will influence cleavage efficiency, we firstly have to take a deep look at the mechanism of 2A cleavage.

The most acceptable model shows 2A mediates a cotranslational “ribosome skipping” event here, it predicts that the nascent 2A interacts with the exit tunnel of the ribosome to affect the conformational space occupied by the ester bond linking the nascent protein and tRNAGly in the ribosome P-site. The model predicts that the residues may influence activity reside within the exit tunnel of the ribosome. Also the structural data shows that the ribosome exit tunnel may accommodate 30-40 amino acids. So the activity of shorter forms of F2A may be affected by the nature of the C-terminal sequence of the protein upstream .Then we use secondary structural prediction program to predict the 2A region structure, the result showed that it will form an a helices capped that their C termini. And this has been more stringently tested by dynamic molecular modeling studies on FMDV 2A by J.Wilkie.

Prediction result from Protein Homology/analogY Recognition Engine V 2.0.

To get the most suitable length of 2A, we analyze the stability of 2A linkers with different lengths. Here we use ProtParam to analyze the 2A sequence listed on the paper to calculate the unstable index. The instability index provides an estimate of the stability of your protein in a test tube. Statistical analysis of 12 unstable and 32 stable proteins has revealed [7] that there are certain dipeptides, the occurrence of which is significantly different in the unstable proteins compared with those in the stable ones. The authors of this method have assigned a weight value of instability to each of the 400 different dipeptides (DIWV). Using these weight values it is possible to compute an instability index (II) which is defined as:

DIWV(x[i]x[i+1]) is the instability weight value for the dipeptide starting in position i.

A protein whose instability index is smaller than 40 is predicted as stable, a value above 40 predicts that the protein may be unstable. The statistical result shows here.

Since 2A with 22 amino acids shows lowest unstable index, it is the most stable one, which will unlikely to be affected by upstream sequences.

So using the results of our pre analysis, we constructed our device and did our experiments.

Our experiment

We want to do more

Bio safety

We have plans to make further modifications to the system, adding a toxic gene in between the two target genes in case of super weed caused by genetic drift. And the application of this vector is restrained within the crops that won't be disturbed by the toxin. In our bettered system, only one herbicide gene is at the risk of drifting because if more genes were to drift into another species, it would definitely include the toxic gene and disrupt the normal life of weeds and thus can't live. The genetic drift rate of each gene is at the end of the day very low, and present prevention methods are proven to be efficient. With our modification, this problem is kept at a even smaller scale.

"Super Crop"

We aims to create a kind of crop that is resistant to different kinds of stress and having other good qualities. Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders. Co-expressing different pesticidal protein in crops using 2A can help create insect-resistant crops.

Also transimitting the LEA protein family can improve the cold-resistant ability of plants. SOS 1,2,3 cooperate to regulate ion homeostasis under salt stress. SOS family is also under consideration to resist salt stress. We also want to transform the metabolic pathways. C4 photosynthesis is a series of anatomical and biochemical modifications to the typical C3 pathway that increases the productivity of plants in warm, sunny, and dry conditions. Co-expressing key enzyme of C4 plant in C3 plant may help to modify the classical C3 pathway.

References

[1]Dill GM, Cajacob CA, Padgette SR. Glyphosate-resistant crops ：adoption, use and future considerations. Pest Manag Sci, 2008, 64（4）:326-331.

[2]Claire Halpin,Susan E. Cooke, Abdellah Barakate,Abdel El Amrani and Martin D. Ryan.Self-processing 2A-polyproteins – a system for co-ordinate expression of multiple proteins in transgenic plants.The Plant Journal (1999) 17(4), 453–459.

[3]Ekterina Minskaia and Martin D.Ryan.Protein Coexpression Using FMDV 2A Effect of "Linker" Residues.BioMed Research International 2013;Article ID 291730

[4]Michelle L. L. Donnelly, Garry Luke,Amit Mehrotra,Xuejun Li,Lorraine E. Hughes,David Gani and Martin D. Ryan.Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’mechanism indicates not a proteolytic reaction, but a novel translational effect : a putative ribosomal ‘skip’.Journal of General Virology (2001), 82, 1013–1025

[5]Ekaterina Minskaia, John Nicholson and Martin D Ryan.Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications.BMC Biotechnology 2013, 13:67

[6]Martin D. Ryan, Michelle Donnelly, Arwel Lewis, Amit P. Mehrotra,John Wilkie, and David Gani.A Model for Nonstoichiometric, Cotranslational Protein Scission in Eukaryotic Ribosomes.Bioorganic Chemistry 27, 55–79 (1999)

[7]Pablo de Felipe, Garry A. Luke, Jeremy D. Brown and Martin D. Ryan.Inhibition of 2A-mediated ‘cleavage’ of certain artificial polyproteins bearing N-terminal signal sequences.Biotechnol. J. 2010, 5, 213–223