— The Value of Altering Plant Phenotypes —
Plant researchers thrive on finding ways to alter a plant's gene expression in order to gain an understanding of the gene's function. This can be done in various ways - by disrupting a gene so that it can't do it's job (creating a mutation); by limiting how much protein a gene can make (influencing gene regulation); or by adding a new gene to the plant.
— How to Alter Phenotypes —
(1) Disrupt a Gene - Making a “Knockout” Mutant
A mutation is any change to the genetic, hereditary structure of an organism. Most mutations negatively affect fitness, some are neutral, and a few provide a selective advantage. Some mutations have no apparent effect on the organism's phenotype - i.e., its appearance, growth or behaviors seem unchanged. These are called “silent” mutations. But many mutations produce an alternate
phenotype -- wrinkled seeds, shorter/taller plants, alterations in flower color - giving researchers a useful way to investigate the underlying function of the responsible gene(s).
For example, by studying mutant maize plants that are less susceptible to ultraviolet radiation (a problem at higher altitudes), researchers can identify genes that cause this advantageous trait. Those genes might then be transferred to other agricultural species grown at high altitudes - providing potential economic benefit to poor communities in the mountainous regions of South America and Asia.
As another example, research with plants incapable of producing vitamin C showed that vitamin C is important in enabling plants to cope with environmental stress, and by deduction, is crucial to humans as well.
One technique for generating plant mutants uses the action of transposons, or jumping genes. These are mobile genetic elements that can excise themselves from DNA and re-insert into another area of the genome. When a transposon inserts into a gene, that gene is no longer capable of producing its associated protein. Its function is “knocked out.” Such knockout mutations can help researchers determine a gene's contribution to discrete phenotypic traits.
Transposons exist naturally in corn and other plants. Indeed, they were originally detected in the 1940s in maize by Barbara McClintock. Since then, researchers have found that they are a useful tool. For example, transposons have been artificially constructed to help locate maize genes for sequencing
McClintock was awarded the 1983 Nobel Prize in Medicine forty years after she started her maize research on transposable DNA. Maize provided the first examples of mobile DNA, which was subsequently shown to be the mechanism permitting rapid movement of antibiotic genes from one bacterium to another - in this example it is the ability of the transposon to excise from one chromosome and move to another that is the key feature. Sometimes, it is the ability of the
transposon to generate allelic diversity that is important: transposons are responsible for the capacity of the human immune system to generate millions of different antibody proteins from just a handful of genes. Two transposons are embedded in the antibody genes, and when they excise individual cells of the immune system end up with slightly different versions of the antibody-encoding genes. When confronted with a new challenge, the few cells
producing antibodies that recognize the problem are selected for rapid growth so that the organism can rid itself of the disease organism or allergen.
— Disrupt Gene Regulation: “Knockdown” Technology —
A cell can regulate gene expression at many steps:
- transcriptionally (how many copies of an mRNA transcript are produced from a gene)
- post-transcriptionally (how many mRNAs are allowed to enter the cytoplasm)
- translationally (how much protein is produced from an mRNA transcript)
- post-translationally (how long the protein is “stable” and whether it has molecular modifications to change how it interacts within the cell)
Antisense transcription and RNA interference (RNAi) regulate gene expression transcriptionally and post-transcriptionally. Plant science researchers have developed tools that boost these regulators in order to observe their effect on a plant's phenotype.
The mRNA strand generally used for translation is termed the sense strand and is transcribed from the antisense DNA strand. Most mRNAs are transcribed in the sense orientation. However, it is also possible for a cell to regulate sense transcription by way of antisense transcripts. These mRNAs, in contrast to their sense counterparts, are transcribed from the DNA sense strand of the same gene. They are therefore complementary to their “sister” sense mRNAs in the cell. The sense and antisense mRNAs come together to form a double-stranded (ds) molecule in the nucleus or cytoplasm. The ds-antisense mRNA
molecule is then cleaved into smaller fragments by an enzyme called Dicer. When combined with a protein complex, these short interfering RNAs (siRNAs) or micro RNAs (miRNAs) can pair with a complementary mRNA molecule and prevent it from being translated into protein. Usually, siRNAs function by degrading the complementary sense strand and miRNAs by blocking translation. These roles are only generally defined, and both siRNA and miRNA have been shown to have similar effects in different organisms, including humans. Much current research focuses on “engineering” siRNAs capable of attacking specific cancer cells.
The ability of antisense RNA to interfere with gene expression was first discovered in an experiment with red petunias in the 1980s. The plant was modified so that the antisense transcript of a gene encoding red flower pigment was expressed in addition to the sense RNA for that gene. The result: a white flower phenotype. Expression of the red pigment protein had been suppressed by the antisense RNA. Like McClintock's work on maize transposons, the petunia research demonstrates the value that plant research has for medicine.
The existence of siRNAs that interfere with gene expression has been exploited in new technologies to control gene expression in transgenic organisms. Scientists have used synthetic versions of antisense mRNA (RNAi) to “silence” gene expression in living plants. This has provided support for both the importance of siRNA and the phenotypic role of the targeted gene. siRNAs usually decrease but do not eliminate expression of the target gene. This phenomenon is referred to as "knockdown" technology.
— Add a New Gene: Introduction of Heterologous Genes —
Another approach to studying altered phenotypes is to introduce a gene with known function from a different organism into a target organism to obtain, usually, a discrete, specifically expressed trait. This strategy has been used to confer pest or herbicide resistance to crop plants. Examples include Bt corn, Bt cotton, and Roundup Ready soybeans.
See: Transgenic Crops Currently on the Market