Plant genetic engineering
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Early research established that the full naturally occurring Bt protoxin is a protein containing 1156 amino acids (later work has shown that insecticidal activity results from a much shorter amino acid sequence).
The Bt protoxin gene was introduced into plant cells using a vector based on the Ti plasmid of Agrobacterium tumefaciens. In the technique used in this case, sometimes called the cointegrate vector technique, all of the sequences are found within a single recombinant Ti plasmid (Figure 1).
A suitable strain of A. tumefaciens containing the recombinant Ti plasmid was generated. Infection of plant tissue with this strain allowed the integration of the protoxin gene into the plant genome.
Figure 1 The recombinant Ti plasmid used to introduce the Bt protoxin gene into plant cells via Agrobacterium tumefaciens mediated transformation. The bacterial selectable marker gene confers resistance to 'antibiotic 1' while the plant selectable marker gene confers resistance to 'antibiotic 2'.
(a) The recombinant Ti plasmid shown in Figure 1 was used to introduce the Bt protoxin gene into plant cells via Agrobacterium tumefaciens mediated transformation.
(i) Explain, in your own words, what the term 'recombinant Ti plasmid' means.
(ii) What is the minimum length of DNA that would be required to code for the full Bt protoxin? If the protoxin had been derived from a plant rather than a bacterium, how would you expect the gene to differ in length? Explain your answer.
(b) Describe briefly the role of the vir region, and explain the significance of its location in the recombinant Ti plasmid.
(c) The plasmid contains two sequences labelled as ORIs and two labelled as selectable marker genes (these are genes for resistance to two different antibiotics - see Figure 1). Explain the roles of these four sequences. You should make it clear why there are two ORIs and two selectable marker genes. Explain why the particular location of the plant selectable marker gene within the recombinant Ti plasmid is crucial, while the precise position of the bacterial selectable marker gene within the plasmid is less important.
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Solution Summary
The Ti plasmid of agrobacterium tumefaciens provides an essential method for genetic engineering of plant cells. This solution provides a detailed explanation of the various elements of a Ti-based vector, and explains their functions and why they are necessary.
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(i) Explain, in your own words, what the term 'recombinant Ti plasmid' means.
The term refers to a plasmid originally derived from Agrobacterium tumefaciens, that has had additional genes or other DNA elements added. The additional sequences derive from other organisms, which is why it is referred to as a recombinant plasmid. In this case there is a plant antibiotic resistance gene, a bacterial antibiotic resistance gene and a sequence from the bacterium E. Coli.
(ii) What is the minimum length of DNA that would be required to code for the full Bt protoxin? If the protoxin had been derived from a plant rather than a bacterium, how would you expect the gene to differ in length? Explain your answer.
As the protein is 1156 amino acids, and each amino acid is encoded by a codon consisting of thee bases, the minimum size of the gene would be 1156 x 3 = 3468 base pairs. Unlike most bacterial genes, plant genes are generally divided into several exons, separated by introns, which extends the total size of the gene.
(b) Describe briefly the role of the vir region, and ...
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