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SSH for revealing functional and immune responses in prawn larvae
Suppression subtractive hybridization (SSH) is a powerful technique to study gene expression in specific tissues, cell types, at a specific life-stage or in a disease-state. It is an ideal tool for identification of differentially expressed genes. It is is a PCR based technique that can be used to to quickly compare the expression of mRNA and show the relative difference in mRNA concentration from two different samples. It is useful to detect alterations in gene expression that is generally associated with a large spectrum of biological and/or pathological processes. Thus, identification of gene expression profiles through subtractive hybridization helps us to understand the molecular mechanisms behind any alteration. The way SSH works is somewhat tricky and, has several procedural steps. The first step being, conversion of mRNA to cDNA in vitro using reverse transcriptase. It then involves a hybridization step between the cDNA of tester population (TP) and driver population (DP). The TP is a set of experimental organisms whose gene expression has been altered by some external stimulus that could be a pathogen, chemical, or any kind of stress. Essentially, the TP will be containing differentially expressed genes. The DP (or, reference population) would be a control set without an exposure to experimental stimulus. The TP cDNA is hybridized with that of DP cDNA and through repeated cycles of PCR using two sets of adaptors ligated, the TP cDNA is quantitatively enhanced. By all these steps, there will be suppression of the genes common to both TP and DP. But, there will be enrichment of differentially expressed genes from TP. Scientists at the NIO adapted the SSH to study the pathogenic effects of Vibrio harveyi, a known bacterial pathogen in marine shrimp hatcheries in the country. The genes upregulated in 15-days old post-larvae of Penaeus monodon (Fig 1) and Penaeus indicus (Fig 2) after exposure to Vibrio harveyi have been identified through other sets of molecular techniques. For this, cDNA libraries were constructed from both the species of prawns. From the 70 cDNA clones sequenced so far, genes related to oxidative stress/ ATP metabolism such as ATP synthase, cytochrome coxidase, NADH dehydrogenase have been observed to be upregulated. Also, immune related genes such as ferritn, metalloproteinase, and peptidases are detected. Several cytoskeletal and protein synthesis related genes appear to be upregulated in the post larvae exposed to this bacterium. Application of SSH technique would lead to a) designing effective strategies in shrimp health management; b) better understanding of disease pathogenesis and in general, the invertebrate immune system.
This work is carried out by Mr. Sagar Nayak under the DBT Project granted to the NIO scientists Dr N Ramaiah and Mr R A Sreepada. Dissertation student Mr K Ajay from Bharathiar University, Coimbatore also participated in this effort.
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