Question

Imagine that you are a scientist that wants to isolate the gene that codes for Succinate Dehydrogenase (an important Citric Acid Cycle enzyme) from a novel plant that produces lettuce. Knowing concepts in Genetics and bringing in biotechnology, how would you go about cloning and further sequencing this gene using Bioinformatics? Please list in sequential order how you would go about going forward with this experiment

Answer 1

Ans: In the case of plants and other eukaryotes, enzyme succinate dehydrogenase is generally located in the inner membrane of mitochondria. It has also been reported from bacteria and lower eukaryotes such as yeast, Saccharomyces cerevisiae. The function of this enzyme is to catalyze the reaction in which succinate is oxidized to fumarate and FAD is converted into FADH2. Its key role has been well recognized in salicylic acid signaling that in turn induce a stress response in the plants. When a researcher working on the novel plant which produces legume and wants to clone and sequence succinate dehydrogenase gene, a combination of wet-lab experiments along with bioinformatic analysis should be an ideal approach. It is admirable that advent in sequencing technology has accelerated bioinformatics based analysis of the plant genomes The reason is pretty clear i.e. both are interdependent and it will help us to confirm our results more profoundly. So the following methodology could be useful in this regard.

A) Isolation of plant genomic DNA

• In this experiment, one needs to isolate plant genomic DNA using standard methodology. The ommercially available plant genomic DNA isolation kits may be useful to achieve best results. Qualitative and quntitative assessment of this genomic DNA should be performed.

B) Characterizing succinate dehydrogenase gene from novel leguminous plant:

• In this experiment, one should design forward and reverse primers specific for bacterial succinate dehydrogenase enzyme that can be utilized further to amplify this gene from the plant derived genomic DNA. It is because this enzyme is pretty much conserved across domains and their evolutionary significance or relationship can be traced by phylogenetic analysis.
• Further, EcoRV restriction enzyme may be useful to find out this enzyme site into the amplified DNA that can carry all subunits of succinate dehydrogenase enzyme.
• It could be further cloned in the specific types of either electrocompetent or chemically competent Escherichia coli cells and further analyzed for the presence of succinate hydrogenated gene (actually it is a combination of four subunits i.e. sdhCDAB, an actual order of the subunits in a gene/nucleotide sequence). The Sanger sequencing methodology may be employed to obtain the nucleotide sequence of the cloned DNA. This cloned gene can be expressed in a compatible expression host and further its recombinant production and purification could be established. The molecular weight may be assessed by electrophoretic techniques and mass spectrometry.

(C) Establishing a relationship between cloned succinate dehydrogenase enzyme and mitochondrial succinate dehydrogenase

• The establishment of phylogenetic relationship between the obtained nucleotide sequence of SDH gene (more correctly amino acid sequence) and those of the mitochondrial SDH gene from the public database could be done by using BLAST analysis (protein BLAST). All publically available SDH sequences may be downloaded from the National Centre for Biotechnology Information (NCBI) website. Also, the plant genome database website also will be useful.
• It would give us information on the level of similarity/identity between the reference and query SDH protein (amino acid sequence) is.
• The reference SDH sequence can be derived from the bacterial genome.
• Monophyletic clade would result in very close phylogenetic relevance. The phylogenetically close cluster formation would be useful to infer the evolutionary relationship between plant as well as bacterial SDH.