13.1 Essential ideas

13.1.2 Biotechnology in agriculture

Isolating target genes to make recombinant DNA

Figure 13.1.2a – Target gene (ORF) and linked sequences of DNAFigure 13.1.2a – Target gene (ORF) and linked sequences of DNA

  • Bioinformatics plays a role in identifying target genes – databases can be used to find ORFs.
  • Many different ORF finding tools are available to researchers and sequences can be compared using other database tools, especially BLAST.

Figure 13.1.2b- Different reading frames of the same DNA sequence. The first reading frame contains the most longest sequence of uninterrupted codons, and is the most “open”.(source: Wikipedia)Figure 13.1.2b- Different reading frames of the same DNA sequence. The first reading frame contains the most longest sequence of uninterrupted codons, and is the most 'open'. (Source: Wikipedia)

  • The ORF (i.e. the DNA sequence of the target gene) is the main component of the recombinant DNA construct.  
  • The target gene is linked to other sequences that control its expression, including the upstream promoter regions and the downstream terminator sequence. These non-coding regions as well as other linked genes also need to be included in the recombinant DNA.

Marker genes

  • Another component of the recombinant DNA are marker genes, which make it possible to distinguish cultures that have been successfully taken up by the target gene.
  • Unlike most target genes, marker genes code for proteins that can be easily detected in the early stages of the development of plant tissue cultures.
  • The gene for green fluorescence protein (GFP) is a commonly used screenable marker.
  • A gene that codes for antibiotic resistance is a commonly used selectable marker:

Figure 13.1.2c – Testing for successful uptake of target geneFigure 13.1.2c – Testing for successful uptake of a target gene

  • When exposed to antibiotics, the shoots on the left grow normally while there is no growth on the right. This indicates that neither the marker gene (antibiotic resistance), nor the target gene have been successfully incorporated into the genome of the cells on the right.

Methods of gene transfer

  • Once the recombinant DNA is made, it must be inserted into the plant cell and taken up by its chromosome or chloroplast DNA.
  • This can be achieved indirectly using viral or bacterial vectors. See 3.1.5 and 13.2.2.
  • Recombinant DNA may be transferred directly into cells by chemical means, including:
    • Liposomes – small membrane-bound structures that encapsulate the recombinant DNA plasmid, then fuse with the plasma membrane of cells to release DNA into the cell.

Figure 13.1.2d – Liposomes fuse with plasma membranes to deposit DNA (source: Cambridge Biology for the IB Diploma, 2014)Figure 13.1.2d – Liposomes fuse with plasma membranes to deposit DNA (Source: Cambridge Biology for the IB Diploma, 2014)

  • Calcium chloride transformation – suspended cells are exposed to recombinant DNA in calcium chloride solution. The precipitate is able to traverse the plasma membrane when a heat shock is applied.

Figure 13.1.2e – Calcium chloride transformation (source: Wikipedia)Figure 13.1.2e – Calcium chloride transformation (Source: Wikipedia)

  • Three important physical methods are:

Figure 13.1.2f – ElectroporationFigure 13.1.2f – Electroporation

  • Electroporation – plants cells are exposed to a temporary electric field that changes the permeability of membranes and allows for DNA to enter.

Figure 13.1.2g – MicroinjectionFigure 13.1.2g – Microinjection

  • Microinjection – a very small needle is used to inject the target genes directly into the plant cells in vitro.

Figure 13.1.2h – Biolistic methodFigure 13.1.2h – Biolistic method

  • Biolistic method DNA molecules are coated in inert nanoparticles and shot directly into plant cells using a 'gene gun'.
  • Note that the recombinant DNA can be introduced into whole plants, leaf discs or protoplasts, which are plant cells that have had their cell walls removed.

Figure 13.1.2i – PlasmidFigure 13.1.2i – Plasmid
Simplified map of recombinant DNA plasmid.

 Key questions

Figure 13.1.2j – Biolistic gunFigure 13.1.2j – Biolistic gun
A “gene gun” can be used to inject DNA into plant tissues.

Figure 13.1.2k – Screenable markerFigure 13.1.2k – Screenable marker
Fluorescence in a transgenic plant leaf indicates successful uptake of another target gene.

Course links

  • Review how bacterial plasmids are used as vectors for gene transfer. See 3.1.5.
  • Recall some functions of non-coding regions of DNA in 7.1.1.
  • Learn more about BLAST in 13.1.5.