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Characterization and expression of daf-9 and daf-12 genes in the pinewood nematode, Bursaphelenchus xylophilus


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Dauer formation is a mechanism by which nematodes cope with difficult environmental circumstances. DAF-encoding genes are known to control the formation of dauer larvae in several nematode species. In this article, two important daf genes were isolated from the invasive plant parasitic nematode, Bursaphelenchus xylophilus, which has two dauer larval stages for overwintering and transmitting by vector beetles in its life cycle. A 1915-bp full-length cDNA of Bx-daf-9 was obtained, containing a 1533-bp open reading frame (ORF), encoding a polypeptide of 510 amino acids. A 3966-bp full-length cDNA of Bx-daf-12 was obtained, containing a 2712-bp ORF, which encoded a polypeptide of 903 amino acids. The structures of both genes in nematodes were compared. A 2758-bp promoter region of Bx-daf-9 and a 2459-bp promoter region of Bx-daf-12 were obtained, with eight putative response elements binding transcription factors in each promoter region. The quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) was performed to detect the mRNA expression levels of the two genes in different stages of the nematode. The result showed that expression levels of the two genes were low in egg stage and up-regulated five to seven times in larval and adult stages. However, Bx-daf-9 expression in the third dispersal stage was relatively low. Expression of daf-12 in the fourth dispersal stage was extraordinarily high. The results imply that daf-9 and daf-12 perhaps have special roles in the dispersal stages of B. xylophilus.