Objective An expansin gene BdEXP1 was cloned from Bursaphelenchus doui, and its expression characteristics were investigated in order to provide reference for the in-depth study of the function of BdEXP1 gene in the interactions between B. doui and hosts.
Method Based on the previous screening and sequencing data of specific cDNA library of the anterior end of B. doui, the full-length cDNA sequence of EXP homologue was cloned by using RACE technology and further analyzed by bioinformatics. The gene copy number was identified by Southern blot. Further, the BdEXP1 expression pattern was investigated by real-time fluorescent quantitative PCR and the expression location was detected by in situ hybridization.
Result The full-length cDNA of BdEXP1 gene was cloned (GenBank accession number: PQ276072). The cDNA coding region was 450 bp encoding 149 amino acids. The DNA coding region was 499 bp containing two exons and one intron. BdEXP1 was a member of a multicopy gene family identified by Southern blot. The BdEXP1 protein had an average molecular mass of 16.16 kD and a theoretical isoelectric point of 7.47. The BdEXP1 protein had the highest sequence similarity (64%) with the homologous protein of B. xylophilus, and contained a typical DPBB_SPI-like conservative domain and a signal peptide but no transmembrane structure, indicating that it was a secretory protein. The result of real-time fluorescent quantitative PCR showed that there were no significant differences in the expression levels of BdEXP1gene in all stages, and in situ hybridization analysis showed that the transcripts of BdEXP1 accumulated exclusively within the esophageal gland of B. doui.
Conclusion The BdEXP1 gene was successfully cloned, and was also found to be expressed within the nematode esophageal gland of B. doui. Therefore, it was speculated that the BdEXP1 protein was excreted out of the body to play a role in interactions between nematode and host.
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