Cloning of Bursaphelenchus xylophilus Pectate Lyase 2 Gene and Construction of Its RNA Interference Vector

QIU Xiu-wen; WU Xiao-qin; HUANG Lin; YE Jian-ren

Volume 29 Issue 2

Article Contents

Turn off MathJax

Article Navigation > Forest Research > 2016 > 29(2): 283-288

Citation:

Cloning of Bursaphelenchus xylophilus Pectate Lyase 2 Gene and Construction of Its RNA Interference Vector

1.
Poyang Lake Eco-economy Research Center, Jiujiang University, Jiujiang 332005, Jiangxi, China
2.
Jiangsu Key Laboratory for Prevention and Management of Invasive Species, College of Forest, Nanjing Forestry University, Nanjing 210037, Jiangsu, China

Received Date: 2015-10-23

Abstract

[Objective]To establish the interference vector of Bxpel2 gene in Bursaphelenchus xylophilus. [Method]Total RNA was extracted and reverse transcribed into cDNA. The primer of Bxpel2 gene containing T7 promoter was designed to amplify the fragment of Bxpel2 gene. The products of amplification were connected to RNAi vector and used to amplify the target fragment of double strand RNA (dsRNA) of Bxpel2 gene. The expression of Bxpel2 gene was tested by quantitative real-time PCR (qRT-PCR). [Result](1) The total RNA extracted by Trizol method was complete and without degradation. (2) The Bxpel2 gene (790bp) of B. xylophilus was cloned and connected to pMD19-T vector. (3) dsRNA was synthesized based on RNA interference vector template, with the concentration of 1.313 and 1.152 mg·mL-1, respectively. (4) The expression of Bxpel2 gene was inhibited by dsRNA interference. [Conclusion]The RNA interference vector of Bxpel2 gene was established successfully. The constructed expression vector could provide a better understanding of the role and function of Bxpel2 gene during the pathogenic process of B. xylophilus.
- Bursaphelenchus xylophilus,
- Bxpel2 gene,
- RNA interference

References

[1]	Zhu L H, Ye J R, Negi S, et al. Pathogenicity of aseptic Bursaphelenchus xylophilus[J]. PLoS ONE, 2012, 7(5): e38095.
[2]	Pereira F, Moreira C, Fonseca L, et al. New insights into the phylogeny and worldwide dispersion of two closely related nematode species, Bursaphelenchus xylophilus and Bursaphelenchus mucronatus[J]. PLoS ONE , 2013, 8(2): e56288.
[3]	Mallez S, Castagnone C, Espada M, et al. First insights into the genetic diversity of the pine wood nematode in its native area using new polymorphic microsatellite loci[J]. PLoS ONE, 2013, 8(3): e59165.
[4]	Zhao H, Chen C, Liu S, et al. Aseptic Bursaphelenchus xylophilus does not reduce the mortality of young pine tree[J]. Forest Pathology, 2013, 43(6):444-454.
[5]	Qiu X W, Wu X Q, Huang L, et al. Specifically Expressed Genes of the Nematode Bursaphelenchus xylophilus involved with early interactions with pine trees[J]. PLoS ONE, 2013, 8(10): e78063.
[6]	Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391 (6669):806-811.
[7]	Elen A F, M Auro D V, John T J, et al. Analysis of chitin synthesis function in a plant parasitic nematode, Meloidogyneartiellia, using RNAi[J].Gene, 2005, 349(2): 87-95.
[8]	Kimber M J, McKinney S, Master S, et al. Flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference[J]. Faseb Journal, 2007, 21(4): 1233-1243.
[9]	Kikuchi T, Shibuya H, Aikawa T, et al. Cloning and characterize attion of pectate l yeasts expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus[J]. Molecular Plant-Microbe Interactions, 2006, 19(3): 280-287.
[10]	Mota M M, Takemoto S, Takeuchi Y, et al. Comparative studies between Portuguese and Japanese isolates of the pinewood nematode, Bursaphelenchus xylophilus[J]. Nematology, 2006, 38(4): 429-433.
[11]	Cheng X Y, Dai S M, Xiao L, et al. Influence of cellulase gene knockdown by dsRNA interference on the development and reproduction of the pine wood nematode, Bursaphelenchus xylophilus[J]. Nematology, 2010, 12(2): 225-233.
[12]	Jackson A L, Linsley P S. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application[J]. Dressnature Reviews Drug Discovery, 2010, 9(1): 57-67.
[13]	Persengiev S P, Zhu X, Green M R. MR: Nonspecific, concentrationdependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs)[J]. Rna-a Publication of the Rna Society, 2004, 10(1): 12-18.
[14]	Caffrey D R, Zhao J, Song Z L, et al.siRNA off-target effects can be reduced at concentrations that match their individual potency[J] . PLoS ONE,2011,6(7) : e21503.
[15]	Ma H B, Lu Q, Liang J, et al. Functional analysis of the cellulose gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference[J]. Genetics & Molecular Research, 2011, 10(3): 1931-41.
[16]	Lee D W, Kang J S, Chan S J, et al. Identification and biochemical analysis of a novel pectate lyase 3 gene in Bursaphelenchus xylophilus[J]. Journal of Asia-Pacific Entomology, 2013, 16(3): 335-342.
[17]	Kulas J, Schmidt C, Rothe M, et al. Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans. Archives of Biochemistry & Biophysics, 2008, 472(1): 65-75.
[18]	Aikawa T, Kikuchi T. Estimation of virulence of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) based on its reproductive ability. Nematology, 2007, 9(3): 371-377.
[19]	Shinya R, Takeuchi Y, Ichimura K, et al. Establishment of a set of inbred strains of the pine wood nematode, Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae), and evidence of their varying levels of virulence. Applied Entomology & Zoology, 2012, 47(4): 341-350.

Proportional views

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Get Citation

PDF

XML

Article views(2809) PDF downloads(801) Cited by()

Proportional views

Cloning of Bursaphelenchus xylophilus Pectate Lyase 2 Gene and Construction of Its RNA Interference Vector

1. Poyang Lake Eco-economy Research Center, Jiujiang University, Jiujiang 332005, Jiangxi, China

2. Jiangsu Key Laboratory for Prevention and Management of Invasive Species, College of Forest, Nanjing Forestry University, Nanjing 210037, Jiangsu, China

Received Date: 2015-10-23

Abstract: [Objective]To establish the interference vector of Bxpel2 gene in Bursaphelenchus xylophilus. [Method]Total RNA was extracted and reverse transcribed into cDNA. The primer of Bxpel2 gene containing T7 promoter was designed to amplify the fragment of Bxpel2 gene. The products of amplification were connected to RNAi vector and used to amplify the target fragment of double strand RNA (dsRNA) of Bxpel2 gene. The expression of Bxpel2 gene was tested by quantitative real-time PCR (qRT-PCR). [Result](1) The total RNA extracted by Trizol method was complete and without degradation. (2) The Bxpel2 gene (790bp) of B. xylophilus was cloned and connected to pMD19-T vector. (3) dsRNA was synthesized based on RNA interference vector template, with the concentration of 1.313 and 1.152 mg·mL-1, respectively. (4) The expression of Bxpel2 gene was inhibited by dsRNA interference. [Conclusion]The RNA interference vector of Bxpel2 gene was established successfully. The constructed expression vector could provide a better understanding of the role and function of Bxpel2 gene during the pathogenic process of B. xylophilus.

HTML

Reference (19)

[1]	Zhu L H, Ye J R, Negi S, et al. Pathogenicity of aseptic Bursaphelenchus xylophilus[J]. PLoS ONE, 2012, 7(5): e38095.
[2]	Pereira F, Moreira C, Fonseca L, et al. New insights into the phylogeny and worldwide dispersion of two closely related nematode species, Bursaphelenchus xylophilus and Bursaphelenchus mucronatus[J]. PLoS ONE , 2013, 8(2): e56288.
[3]	Mallez S, Castagnone C, Espada M, et al. First insights into the genetic diversity of the pine wood nematode in its native area using new polymorphic microsatellite loci[J]. PLoS ONE, 2013, 8(3): e59165.
[4]	Zhao H, Chen C, Liu S, et al. Aseptic Bursaphelenchus xylophilus does not reduce the mortality of young pine tree[J]. Forest Pathology, 2013, 43(6):444-454.
[5]	Qiu X W, Wu X Q, Huang L, et al. Specifically Expressed Genes of the Nematode Bursaphelenchus xylophilus involved with early interactions with pine trees[J]. PLoS ONE, 2013, 8(10): e78063.
[6]	Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391 (6669):806-811.
[7]	Elen A F, M Auro D V, John T J, et al. Analysis of chitin synthesis function in a plant parasitic nematode, Meloidogyneartiellia, using RNAi[J].Gene, 2005, 349(2): 87-95.
[8]	Kimber M J, McKinney S, Master S, et al. Flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference[J]. Faseb Journal, 2007, 21(4): 1233-1243.
[9]	Kikuchi T, Shibuya H, Aikawa T, et al. Cloning and characterize attion of pectate l yeasts expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus[J]. Molecular Plant-Microbe Interactions, 2006, 19(3): 280-287.
[10]	Mota M M, Takemoto S, Takeuchi Y, et al. Comparative studies between Portuguese and Japanese isolates of the pinewood nematode, Bursaphelenchus xylophilus[J]. Nematology, 2006, 38(4): 429-433.
[11]	Cheng X Y, Dai S M, Xiao L, et al. Influence of cellulase gene knockdown by dsRNA interference on the development and reproduction of the pine wood nematode, Bursaphelenchus xylophilus[J]. Nematology, 2010, 12(2): 225-233.
[12]	Jackson A L, Linsley P S. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application[J]. Dressnature Reviews Drug Discovery, 2010, 9(1): 57-67.
[13]	Persengiev S P, Zhu X, Green M R. MR: Nonspecific, concentrationdependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs)[J]. Rna-a Publication of the Rna Society, 2004, 10(1): 12-18.
[14]	Caffrey D R, Zhao J, Song Z L, et al.siRNA off-target effects can be reduced at concentrations that match their individual potency[J] . PLoS ONE,2011,6(7) : e21503.
[15]	Ma H B, Lu Q, Liang J, et al. Functional analysis of the cellulose gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference[J]. Genetics & Molecular Research, 2011, 10(3): 1931-41.
[16]	Lee D W, Kang J S, Chan S J, et al. Identification and biochemical analysis of a novel pectate lyase 3 gene in Bursaphelenchus xylophilus[J]. Journal of Asia-Pacific Entomology, 2013, 16(3): 335-342.
[17]	Kulas J, Schmidt C, Rothe M, et al. Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans. Archives of Biochemistry & Biophysics, 2008, 472(1): 65-75.
[18]	Aikawa T, Kikuchi T. Estimation of virulence of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) based on its reproductive ability. Nematology, 2007, 9(3): 371-377.
[19]	Shinya R, Takeuchi Y, Ichimura K, et al. Establishment of a set of inbred strains of the pine wood nematode, Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae), and evidence of their varying levels of virulence. Applied Entomology & Zoology, 2012, 47(4): 341-350.

Cloning of Bursaphelenchus xylophilus Pectate Lyase 2 Gene and Construction of Its RNA Interference Vector

Abstract

References

Proportional views

通讯作者: 陈斌, bchen63@163.com

Proportional views

Cloning of Bursaphelenchus xylophilus Pectate Lyase 2 Gene and Construction of Its RNA Interference Vector

HTML

Catalog

Export File

Citation

Format

Content