John Gray is Using Molecular Genetics to Improve Crops

My interest in science derives from my curiosity about nature. That love and curiosity started in Ireland where I was born, and I grew up close to the countryside and the seaside. My parents often took us for walks in the country and vacation by the sea. I started collecting shells before I was 10 years old and identified them using nature guides from the library or as birthday gifts from my my parents. I was also influenced by the Irish wildlife documentaries on TV pioneered by Eamon De Buitléar as well as nature programs by David Bellamy, David Attenborough, and Jacques Cousteau.

What we learn about the regulation of phenylpropanoid genes in maize has relevance in all other plants as the pathway is highly conserved in land plants.

Within cereal plants alone there is a need to breed more robust varieties that can better withstand biotic and abiotic stresses. Often changing the regulation of stress response genes is sufficient to achieve such aims without modifying the genes themselves.

In this sense our findings are expected to be of relevance to plant biotech companies and breeders that are developing cereals that have improved plant stature (i.e. lignin content in cell walls), and biotic or abiotic stress resistance (i.e. disease resistance).

What happens next in the process of discovery?

Next steps in the process of discovery include evaluating key TFs and the mechanism by which they turn on or off target phenylpropanoid genes inside plants. TFs are like keys that turn on and off genes, however they work in dynamic complexes and may have a positive or negative effect on the target gene expression.

We need to fully understand how such complexes work in order to intelligently modify target gene expression. We also envisage identifying corresponding TFs in closely related species such as rice, sorghum and other important cereal crops and understanding how variation in those TFs affects each species. Our TFome can also be applied to understanding the regulation of other genes and pathways in corn and is available to other researchers for such purposes.

Looking toward the future and the next steps in your research, what are you most excited about?

When our findings are combined with new technologies such as gene editing I look forward to seeing the application of our findings to the generation of improved crop varieties. In several ways we are just at the beginning of what can be accomplished in generating improved crop varieties. In the undergraduate molecular genetics laboratory that I teach at UT (BIOL 3020), I challenge students to work in groups to think of how gene editing can be used to improve domesticated species. Every semester I am surprised at the projects they come up with and I am gratified to see some of the same ideas being pursued in biotech companies.

When did you first start using Lasergene?

I have been using Lasergene since the mid 1990’s when I had to analyze DNA sequences that I generated by manual dideoxy sequencing. The SeqMan software greatly eased the process of identifying overlapping reads and identifying sequencing errors (e.g. viewing the underlying chromatograms is very helpful in deciding sequence quality). When we cloned genes from maize the SeqBuilder software made it much easier to identify coding versus non-coding regions and then build and draw accurate gene maps. Once we identified coding regions then of course we needed to compare with homologous sequence from GenBank. Downloading sequences directly from GenBank into Seqbuilder made these studies more straightforward. Then it was easy to compare them using MegAlign and this was useful in identifying conserved regions as well as those unique regions that could be used in antiserum generation. When we worked on generating the maize TFome collection the Lasergene software also proved most useful from primer design to confirming the final clones by sequencing. The interactive primer design interface was very helpful in designing optimal primers when the location was restricted to the beginning and end of the coding region. I witnessed these various Lasergene programs improving over the years and the constant attention to improvement was one of the reasons I stuck with Lasergene over other programs in my research laboratory.

We’ve heard you use Lasergene in a lab class. Tell us more!

In the Molecular Genetics Laboratory (BIOL 3020) that I teach, I employ Lasergene for teaching students to examine genes and their products. The software easily allows first time students to examine DNA and protein sequences and understand gene maps imported from GenBank. The virtual gel tool is very useful when teaching gel electrophoresis, the translation tool allows them to see the consequences of viral genome evolution (i.e. amino acid changes in different coronavirus variants). When we teach comparative genomics, students use MegAlign Pro to make alignments and phylogenetic trees. When they do a virtual gene editing (CRISPR) project they design gRNA/repair template plasmids also using Seqbuilder Pro. All in all, the Lasergene package is most suitable for enabling students to quickly dive into the world of molecular genetics and helps bring sequences alive and more meaningful to them.

I have recruited undergraduate students from BIOL 3020 to further assist me in cloning TF sequences from maize for our TFome project. Having already learned to use Seqbuilder Pro in class they quickly draw all their plasmids and then use SeqMan Pro to check that the cloned inserts are correct. Graduate students also do all their primer design using Seqbuilder Pro and for drawing all Gateway recombinant destination clones. Lastly we have used MegAlign Pro for doing phylogenetic studies – mainly using the CLUSTAL or MUSCLE algorithms contained therein. One of my recent undergraduates was a co-author on a paper because of the insightful phylogenetic trees he generated as part of his honors thesis.

Any last words about Lasergene?

Overall I find the Lasergene package to be most suitable for work in my research lab and for educational purposes. Earlier on we had looked at Vector NTI software (now discontinued) but Lasergene was always more polished and updated. I suspect this is in part because staff at DNASTAR listen to the feedback that is provided to them. Their support has always been helpful to me whenever I called. Over the years I and others have suggested several feature improvements and I was happy to see such improvements appear in subsequent updates. I am confident that DNASTAR will continue to provide such improvements in the future. I also envisage that maybe there will be a day when even the public will have a DNA software package on their laptops to help them understand their own genes and their own health.