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

Sheng Dongping; Zhang Xiaoyang; Feng Mengting; Ye Jianren; Qiu Xiuwen

doi:10.12171/j.1000-1522.20200025

Journal of Beijing Forestry University > 2021 > 43(1): 96-102. > DOI: 10.12171/j.1000-1522.20200025

Sheng Dongping, Zhang Xiaoyang, Feng Mengting, Ye Jianren, Qiu Xiuwen. Construction and its function analysis of RNA interference vector of cytochrome cyp-13A11 gene in Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2021, 43(1): 96-102. DOI: 10.12171/j.1000-1522.20200025

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

Sheng Dongping^{1, 2,},
Zhang Xiaoyang^{1, 2},
Feng Mengting^{1, 2},
Ye Jianren³,
Qiu Xiuwen^{1, 2, ,}

1.
Jiangxi Yangtze River Economic Zone Research Institute of Jiujiang University, Jiujiang 332005, Jiangxi, China
2.
College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China
3.
Jiangsu Key Laboratory for Prevention and Management of Invasive Specie, College ofForest, Nanjing Forestry University, Nanjing 210037, Jiangsu, China

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Received Date: January 19, 2020
Revised Date: April 08, 2020
Available Online: November 23, 2020
Published Date: February 04, 2021

Graphical Abstract

Abstract

Abstract

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.
- Bursaphelenchus xylophilus,
- cyp-13A11 gene,
- vector construction,
- RNA interference,
- function analysis

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References

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

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