Cloning and expression localization analysis of a novel transthyretin-like gene（Bd-TTL-1）from Bursaphelenchus doui

【Objective】A transthyretin-like（TTL）gene Bd-TTL-1 was cloned from Bursaphelenchus doui，and its expression characteristics were investigated in order to lay a foundation for further studying the biology function of Bd-TTL-1 gene.【Method】The plant parasitic nematode Bursaphelenchus doui was used as the test material，the anterior end specific cDNA library was constructed by solid-phase subtractive hybridization using the anterior end cDNA as the test and the posterior end cDNA as the driver. The cDNA library was screened by reverse-northern blotting and sequencing. The full-length cDNA sequence of Bd-TTL-1 gene was obtained by using RACE technology. Bioinformatics software and database were used to analyze the physicochemical properties，signal peptides，transmembrane structures，homology and conserved domains of the Bd-TTL-1 protein. Real-time fluorescence quantitative PCR and in situ hybridization were used to analyze the gene development pattern and expression location.【Result】A specific cDNA library of the anterior end of Bursaphelenchus doui was constructed successfully. Full-length TTL gene Bd-TTL-1 from Bursaphelenchus doui was cloned by using reverse Northern blotting，sequencing and RACE technologies，and its GenBank accession number was PQ276066. The DNA coding region of Bd-TTL-1 gene was 497 bp with 2 exons and 1 intron. The cDNA coding region was 435 bp in length，encoded 144 amino acids. The predicted relative molecular weight of Bd-TTL-1 protein was 15.86 kD，and the theoretical isoelectric point was 9.37. Bd-TTL-1 protein contained a typical and conserved TTL protein domain，with signal peptide but no transmembrane domain，and belonged to the secreted protein. Real-time fluorescence quantitative PCR results showed that Bd-TTL-1 gene was expressed in all development stages of Bursaphelenchus doui， but expression was weak in egg stage，and was significantly lower than other development stages（P<0.05）. The results of in situ hybridization showed that Bd-TTL-1 gene was specifically expressed in the esophageal gland of Bursaphelenchus doui.【Conclusion】The Bd-TTL-1 protein of Bursaphelenchus doui has a typical TTL protein conservative domain，and is a secretory protein with a signal peptide but no transmembrane structure. The Bd-TTL-1 gene is specifically expressed in the esophageal glands of Bursaphelenchus doui，and it is speculated that its coding protein was excreted outside of the body through the stylet to play a role during the parasitism.