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Suppression of Pheromone Biosynthesis Via RNA Interference
Sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN) in many lepidopteran species. A cDNA isolated from female adult heads of Plutella xylostella encodes 193 amino acids including PBAN, designated as Plx-PBAN. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in all examined body parts, with the highest expression level in the head of female adults. The PBAN receptor (Plx-PBANr) gene was also cloned from the female pheromone gland and has conserved structural motifs implicating in promoting G protein coupling and tyrosine-based sorting signaling along with seven transmembrane domains. The expression of Plx-PBANr was found only in the pheromone gland of female adults among examined tissues and developmental stages. Heterologous expression in human uterus cervical cancer cells revealed that Plx-PBANr induced significant calcium elevation when challenged with Plx-PBAN. Female P. xylostella injected with double-stranded RNA specific to Plx-PBANr showed suppression of the receptor gene expression and exhibited significant reduction in pheromone biosynthesis, which resulted in loss of male attractiveness. In addition, to assess molecular events occurring downstream of PBAN signaling, partial sequences of Δ9 and Δ11 fatty acid desaturases of P. xylostella. were cloned. Phylogenetic analysis indicated that these two desaturase genes were highly clustered with other desaturases associated with sex pheromone biosynthesis in other insects. RT-PCR analysis showed that Δ9 desaturase was dominantly expressed in adult females, whereas Δ11 desaturase was expressed in all developmental stages. When PBANr expression was suppressed by PBANr-RNAi, the treated females also showed significant suppression of expression of both desaturases. These results suggest that expressions of the two desaturases are controlled by PBAN and that the two desaturases may be involved as downstream components in sex pheromone biosynthesis of P. xylostella.
