Construction and its function analysis of RNA interference vector of cytochrome cyp-13A11 gene in Bursaphelenchus xylophilus

  Objective  In order to reveal the function of cyp-13A11 gene in Bursaphelenchus xylophilus, the RNA interference vector was constructed and the function of cyp-13A11 gene of Bursaphelenchus xylophilus was analyzed to provide theoretical basis for biological control of pine wilt diseases.

  Method  Specific primers were designed to amplify the cyp-13A11 gene fragment of Bursaphelenchus xylophilus. The products of amplification were constructed into pEASY-T1 interference vector, which was further transferred into Trans1-T1 escherichia coli strain. RNA interference efficiency was determined by qRT-PCR. The feeding reproduction and pathogenicity of pine wood nematodes were investigated after the nematodes soaked in cyp-13A11 dsRNA solution were inoculated on Botrytis cinerea and pine trees, respectively.

  Result  Trans1-T1 strain with pEASY-T1 interference vector was successfully constructed and cyp-13A11 dsRNA was synthesized. RNA interference inhibited the expression of cyp-13A11 gene in Bursaphelenchus xylophilus. In addition, the feeding area was less in cyp-13A11 dsRNA treatment than that in ddH₂O treatment, as two thirds of the Botrytis cinerea were fed by nematodes in ddH₂O treatment on day 3. After 6 days, one third of Botrytis cinerea was fed by nematodes in cyp-13A11 dsRNA treatment, while almost all of the Botrytis cinerea were exhausted by nematodes in ddH₂O treatment. The reproduction number of nematodes in ddH₂O treatment was 4.28 times higher than that in cyp-13A11 dsRNA treatment. Furthermore, the wilting rates in both ddH₂O and cyp-13A11 dsRNA treatments were 22.2% and 5.6% after Bursaphelenchus xylophilus being inoculated on pine trees for 10 days, respectively. The wilting rates of pine trees in both ddH₂O and cyp-13A11 dsRNA treatments were 44.4% and 33.3% after 20 days, respectively. Obviously, the wilting rates were 100% in both treatments after 30 days.

  Conclusion  The RNA interference vector of cyp-13A11 gene in pine wood nematode was successfully constructed. Silencing of cyp-13A11 gene exhibited an important effect on feeding of Bursaphelenchus xylophilus, as well as reduced the reproduction ability and pathogenicity of Bursaphelenchus xylophilus.