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Differentially expressed genes in hemocytes of Vibrio harveyi-challenged shrimp Penaeus monodon
Kunlaya Somboonwiwat1, Premruethai Supungul, Vichien Rimphanitchayakit
1Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Insights
Differential Display PCR identified key genes in shrimp immune response to Vibrio harveyi infection. Several genes, including glucose transporter 1 and lysozyme, showed increased expression, highlighting their role in bacterial defense.
Area of Science:
- Marine Biology
- Immunology
- Molecular Biology
Background:
- Vibrio harveyi is a significant pathogen affecting penaeid shrimp.
- Understanding shrimp immune responses is crucial for aquaculture sustainability.
Purpose of the Study:
- To identify differentially expressed genes in Penaeus monodon hemocytes upon Vibrio harveyi infection using Differential Display PCR (DD-PCR).
- To confirm the expression patterns of selected genes involved in the immune response.
Main Methods:
- Differential Display PCR (DD-PCR) was employed to screen for gene expression changes.
- Sequencing and BLAST analysis were used to identify differentially expressed gene fragments.
- Real-time PCR was utilized to validate the expression levels of selected genes.
Main Results:
- 79 differentially expressed bands were detected, with 48 showing increased and 31 decreased expression.
- Sequence analysis identified 24 known genes, including glucose transporter 1, lysozyme, and SERPINB3, among the differentially expressed fragments.
- Real-time PCR confirmed the up-regulation of selected genes, such as glucose transporter 1 and lysozyme, in response to V. harveyi challenge.
- The anti-lipopolysaccharide factor type 3 (ALFPm3) gene was found to be up-regulated, with observed amino acid diversity.
Conclusions:
- The study identified several genes involved in the shrimp immune response to bacterial infection.
- The findings suggest that genes like glucose transporter 1, lysozyme, and ALFPm3 play a role in defending Penaeus monodon against Vibrio harveyi.
Abstract:
Differential Display PCR technique (DD-PCR) was used for the analysis of altered gene expression in hemocytes of Vibrio harveyi-infected Penaeus monodon. Forty-four combinations of arbitrary and oligo(dT) primers were used to screen for differentially expressed genes. A total of 79 differentially expressed bands could be identified from 33 primer combinations. These included 48 bands (61%) whose expression level increased and 31 bands (39%) decreased after V. harveyi challenge. Subsequently, forty-eight differential display fragments were successfully reamplified and cloned. A total of 267 clones were randomly selected and sequenced. The sequence analysis showed that 85 (31%) out of 267 clones were matched with sequences in the GenBank database which represented 24 different genes with known functions. Among the known genes, glucose transporter 1, interferon-related developmental regulator 1, lysozyme, profilin, SERPINB3, were selected for further confirmation of their differentially expression patterns by real-time PCR. The results showed increasing in expression level of the selected genes in shrimp hemocytes after microbial challenge suggesting the involvement of such genes in bacterial response in shrimp. The anti-lipopolysaccharide factor type 3 (ALFPm3) gene, previously reported in P. monodon (Supungul et al., 2002) was found among the up-regulated genes but diversity due to amino acid changes was observed. Increase in ALFPm3 transcripts upon V. harveyi injection is in accordance with that found in the previous study.
