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CONSTRUCTION OF SILKWORM MIDGUT cDNA LIBRARY FOR SCREEN AND SEQUENCE ANALYSIS OF PERITROPHIC MEMBRANE PROTEIN GENES
Yi-Jun Zhou1, Bin Xue1, Yang-Yang Li1
1School of Basic Medicine and Biological Sciences, Soochow University, Suzhou, Jiangsu, P.R. China.
Abstract:
Silkworm is an important economic insect and the model species for Lepidoptera. The midgut of silkworm is an important physiological barrier, as its peritrophic membrane (PM) can resist pathogen invasion. In this study, a silkworm midgut cDNA library was constructed in order to identify silkworm PM genes. The capacity of the initial library was 6.92 × 10(6) pfu/ml, along with a recombination rate of 92.14% and a postamplification titer of 4.10 × 10(9) pfu/ml. Three silkworm PM protein genes were obtained by immunoscreening, two of which were chitin-binding protein (CBP) genes and one of which was a chitin deacetylase (CDA) gene as revealed by sequence analysis. Three genes were named BmCBP02, BmCBP13, and BmCDA17, and their ORF sizes are 678, 1,029, and 645 bp, respectively; all of them contain sequences of chitin-binding domains. Phylogenetic analysis indicated that BmCBP02 has the highest consensus with Mamestra configurata CBP at 61.0%; BmCBP13 has the highest consensus with Loxostege sticticalis PM CBP at 53.35%; BmCDA17 has the highest consensus with Helicoverpa armigera CDA5a at 70.83%. Tissue transcriptional analysis revealed that all three genes were specifically expressed in the midgut, and during the developmental process of fifth-instar silkworms, the transcription of all the genes showed an upward trend. This study laid a foundation for further studies on the functions of silkworm PM genes.
Insights
Researchers identified key genes in the silkworm midgut peritrophic membrane (PM), crucial for resisting pathogens. This study isolated two chitin-binding protein (CBP) genes and one chitin deacetylase (CDA) gene, advancing understanding of insect immunity.
Area of Science:
- * Entomology
- * Molecular Biology
- * Insect Physiology
Background:
- * Silkworms are vital economic insects and model organisms for Lepidoptera research.
- * The silkworm midgut's peritrophic membrane (PM) acts as a critical barrier against pathogen invasion.
- * Identifying genes within the PM is essential for understanding its protective functions.
Purpose of the Study:
- * To construct a silkworm midgut cDNA library for identifying PM genes.
- * To isolate and characterize novel genes involved in the silkworm's peritrophic membrane structure and function.
Main Methods:
- * Construction of a silkworm midgut cDNA library.
- * Immunoscreening to identify target genes.
- * Sequence analysis to determine gene identity and characteristics (e.g., Open Reading Frame size, chitin-binding domains).
- * Phylogenetic analysis to compare identified genes with known homologs.
- * Tissue transcriptional analysis to assess gene expression patterns.
Main Results:
- * A silkworm midgut cDNA library was successfully constructed with high capacity and recombination rate.
- * Three silkworm PM protein genes were identified: two chitin-binding protein (CBP) genes (BmCBP02, BmCBP13) and one chitin deacetylase (CDA) gene (BmCDA17).
- * Phylogenetic analysis revealed varying degrees of consensus with homologous genes from other insect species.
- * Transcriptional analysis confirmed specific expression of all three genes in the midgut, with increasing trends during the fifth instar developmental stage.
Conclusions:
- * The study successfully identified and characterized three novel silkworm PM genes (BmCBP02, BmCBP13, BmCDA17).
- * These genes are specifically expressed in the midgut and play a role in the peritrophic membrane.
- * This research provides a foundational understanding for future investigations into the functional roles of silkworm PM genes in insect immunity and development.
