Plants are sedentary in that they cannot move around looking for better conditions. However they are able to tolerate stress by producing metabolites, i.e. - Arabidopsis thaliana (a dicotyledon), accumulates the amino acid proline to high levels when exposed to drought stress. The interest therefore is to isolate and characterise the genes of interest that encodes for the production of metabolites that will make the plant to be able to produce whichever metabolites are targeted by the researcher. A. thaliana will be used as a model species in this article.
To be able to isolate a gene mRNA of interest, the mRNA has to be isolated and then be reverse transcribed to cDNA from which the gene can be located. The procedure is as follows:
A healthy leaf or any tissue of A. thaliana where the gene has been shown to be expressed is cut off and crushed in liquid nitrogen using a pestle and mortar. Cetyltrimethylammonium bromide (CTAB), a detergent, is added to the cell extract. CTAB forms an insoluble complex with nucleic acids which precipitates when centrifuged leaving a supernatant that contains carbohydrates, proteins other contaminants that is discarded. The precipitate is resuspended in 1M NaCl which breaks the nucleic acid-CTAB complex and nucleic acids are concentrated by ethanol precipitation. Deoxyribonuclease (DNAse) is then added to degrade DNA leaving behind pure RNA.
DNA-RNA transcription produces mRNA with a poly-A tail. Thus to extract the mRNA, poly-T oligonucleotides are made, attached to glass beads and added to a column. The RNA extract above is then poured into the column and poly-A tail of mRNA hybridizes to the poly-T oligonucleotides in the column and thus it is left behind when all the other RNAs (tRNA and rRNA) leaves the column in solution. The mRNA is then reverse transcribed to form cDNA by adding reverse transcriptase and the cDNA strand replicates to form dsDNA.DNA Polymerase is added to synthesise the cDNA strand complementary to the mRNA template.
The cDNA is then cloned into an expression vector, a plasmid, which is then used to transform bacteria. Therefore the cloned plasmid has a functional gene and this can be used for direct selection. In this instance an Escherichia coli mutant that has a non-functional gene is transformed using the cloned plasmid. The transformed E.coli which carries a plasmid with a functional gene survives in a minimal medium (medium that lacks supplements, e.g. the protein that is encoded for by the gene of interest) and the untransformed E.coli will not survive. Thus the colonies formed in the minimal medium are all recombinants. The exact position of the gene in the recombinant DNA molecule can then be detected, expression levels be determined and translation be verified. This can be done using blotting techniques and hybridization.
Blotting techniques and Hybridisation
The DNA is digested using restriction endonucleases. Then the DNA molecules are isolated using agarose gel electrophoresis. The DNA bands are then transferred from the gel to a nylon membrane. A labelled probe is then applied to locate the DNA molecule or segment that has the gene of interest. The hybridizing probe is synthesised also by transferring DNA molecules to a nylon membrane and then binding the DNA strands to the membrane using ultraviolet irradiation. The probe is then labelled after, removing unbound molecules, using radioactive nucleotides either by nick translation or end filling.
By looking at the Southern hybridisation results, copy number of the gene can also be determined. The gene can also be quantified to see how much of it is expressed depending on conditions that the plant is exposed to. This can be done by Northern blotting where the above mentioned steps as in Southern blotting are used, but here ,mRNA is used and not DNA. Also Real Time Polymerase Chain Reaction (RT-PCR) can be used looking at the amplification on mRNA over time during the reaction. Western bolt can also be used to ascertain the presence of the protein and here antibodies are used as the probe.
After knowing the exact location of the gene, the gene can now be sequenced. This can be done using the Sanger method or Maxam-Gilbert method. But nowadays there are automated DNA sequencing methods that can be used referred to as next generation sequencing techniques such as 454 pyrosequencing, can be used.
During DNA-RNA transcription, non coding sequences (introns) of the DNA are spliced out and only the coding regions (exons) are retained. Thus when sequencing the gene, only the exon sequences can be determined but the intron sequences will not be sequenced. The intron sequence can be determined by comparing the cDNA sequence to the gene sequence of the species. In this case this is possible as A.thaliana genome has been sequenced and thus by base pairing cDNA strand with a complementary strand from the genomic library, intron locations can be seen (whilst observing under the microscope) as they loop out. By comparing cDNA sequence to DNA in genomic library of A .thaliana, the promoter sequence or promoter region of the gene can also be determined. This is done by looking for the TATA box that is immediately close to the gene sequence.
After the gene has been characterised, it is then used for plant transformation, to give the transformed plant the particular phenotype of interest which can be anything from being drought resistant, salinity resistant, insecticides resistance etc. Different transformation techniques can be used depending on which one is more suitable, for instance Agrobacterium-mediated transformation for dicotyledonous plants like A. thaliana, or particle bombardment for most monocotyledonuos plants like rice.
About Author / Additional Info: