Cloning and Analysis of a FMRFamide-like Neuropeptide Gene from Bursaphelenchus doui

doi:10.7668/hbnxb.20195409

Abstract

Abstract:

In order to explore the molecular mechanism of the interaction between Bursaphelenchus doui and host plants,an FMRFamide-like neuropeptide gene,named Bd-FLP-12,was cloned from B.doui by using RACE-PCR technique in a specific cDNA library of the anterior end of B.doui.Further,the nucleotide sequence and amino acid sequence of Bd-FLP-12 were analyzed using bioinformatics methods,the gene copy number was identified by Southern Blot,and the developmental expression pattern was investigated by semi-quantitative RT-PCR.The Bd-FLP-12 gene encoded a protein of 90 amino acids with a mature peptide sequence of FLP-12 and a signal peptide sequence but no transmembrane structure,indicating that its encoded protein was a secretory protein.Southern Blot indicated that the Bd-FLP-12 gene was a single copy gene in the B.doui genome,and the RT-PCR result showed that the transcription level of Bd-FLP-12 was lower in the egg stage than in the other stages.In summary,the full-length sequence of the Bd-FLP-12 gene was cloned for the first time in B.doui,and the structure,nature and expression of the gene were characterized,which could serve as a foundation for further study of the gene function.

Key words: Bursaphelenchus doui, FMRFamide-like neuropeptide, Bioinformatics, Southern Blot, Expression analysis

CLC Number:

S432.45
S763

LIN Shifeng, WANG Rengang, WANG Zili, LI Li, CHEN Yuenan, WU Shasha, LIU Guoqin. Cloning and Analysis of a FMRFamide-like Neuropeptide Gene from Bursaphelenchus doui[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 166-172. doi: 10.7668/hbnxb.20195409.

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Figures/Tables 8

Fig.1 PCR analysis of hsp90 gene using the various round of subtractive hybridization as template M.DL2000 Marker;1.The PCR products of unhybridized cDNA;2.The PCR products of cDNA from the first subtractive hybridization;3.The PCR products of cDNA from the second subtractive hybridization.

Fig.2 Reverse Northern blot analysis using the anterior end and posterior end cDNAs as probes A.The anterior end cDNA probe;B.The posterior end cDNA probe.The single colony numbers are represented by combinations of letters and numbers,where letters represent columns and numbers represent rows.

Fig.3 5' RACE products of the fragment of Bd-m36 M.DL2000 Marker;1.The first PCR product;2.The second PCR product.

Fig.4 Complete cDNA(A)and genomic DNA(B) sequences of the Bd-FLP-12 gene obtained by cloning A.The noncoding regions are indicated with lowercase characters;the coding region and predicted amino acid sequence are indicated with uppercase characters; the stop codon(TAA)is marked with an asterisk;the signal peptide is boxed;the predicted mature peptide RNKFEFIRFamide is marked with a black shadow;the polyadenylation signal sequence(AATAAA)is underlined.B.The boxes represent extrons and the line represents the intron;the first two bases of the intron are GT and the last two bases of the intron are AG;the numbers represent the length of the corresponding sequence.

Fig.5 Southern Blot analysis of Bd-FLP-12 M.The migration positions of the DNA size standards(kb)in agarose gel;N.Genomic DNA of the nematode B.doui;F.Genomic DNA of the fungus B.cinerea;1.Genomic DNA was digested with BamHⅠ;2.Genomic DNA was digested with Hind Ⅲ.

Fig.6 Predicted signal peptide(A)and transmembrane domain(B)of the B.doui Bd-FLP-12

Fig.7 Amino acid sequence alignment of the B.doui Bd-FLP-12 protein with homologues from other plant parasitic nematodes 1.B.doui(Bd-FLP-12); 2.B.xylophilus(ABR09285); 3.A.besseyi(KAI6187848); 4.C.nassatus(CAJ0609964); 5.S.hermaphroditum(KAK0396706); 6.O.dentatum(KHJ99768); 7.M.minor(ABB49052); 8.H.avenae(AEN71840); 9.M.incognita(AAX19364); 10.G.pallida(CAC32452);RNKFEFIRF.Amino-acid sequences of typical petides of FLP-12.

Fig.8 Analysis of the developmental expression pattern of Bd-FLP-12 by semi-quantitative RT-PCR 1.Egg;2.Second-stage juvenile;3.Third-stage juvenile;4.Fourth-stage juvenile;5.Adult female;6.Adult male.

References 28

[1]	Zhuo K, Luo M, Cui R Q, Liao J L. A multiplex one-step PCR method for the simultaneous identification of Bursaphelenchus xylophilus,B.mucronatus and B.doui-three species within the xylophilus group[J]. Forest Pathology, 2011, 41(1):66-69.doi:10.1111/j.1439-0329.2009.00638.x.
[2]	严正梅, 谈家金. 我国松树体内线虫种类及其分布[J]. 中国森林病虫, 2019, 38(5):38-42.doi:10.19688/j.cnki.issn1671-0886.20190015.
	Yan Z M, Tan J Z. Species and geographical distribution of parasitic nematode in pine in China[J]. Forest Pest and Disease, 2019, 38(5):38-42.
[3]	陈耀明. 我国松树体内线虫种类及其分布规律研究[J]. 种子科技, 2019, 37(7):129.doi:10.3969/j.issn.1005-2690.2019.07.110.
	Chen Y M. Species and distribution laws of parasitic nematode in pine in China[J]. Seed Scicence & Technology, 2019, 37(7):129.
[4]	Braasch H, Gu J F, Burgermeister W. Bursaphelenchus doui sp.n.(Nematoda:Parasitaphelenchidae)in packaging wood from Taiwan and South Korea-a new species of the xylophilus group[J]. Russian Journal of Nematology, 2004, 12(2):19-27.
[5]	顾建锋, 张慧丽, 郑炜. 进境木质包装中豆伞滑刃线虫的鉴定[J]. 植物检疫, 2007, 21(3):145-148.doi:10.3969/j.issn.1005-2755.2007.03.005.
	Gu J F, Zhang H L, Zheng W. Identification of Bursaphelenchus doui Braasch,Gu,Brugermeister and Zhang,2004 from imported wooden package in Ningbo Port[J]. Plant Quarantine, 2007, 21(3):145-148.
[6]	Zhou Q Z, Liu Y, Wang Z, Wang H M, Zhang X Y, Lu Q. Rapid on-site detection of the Bursaphelenchus xylophilus using recombinase polymerase amplification combined with lateral flow dipstick that eliminates interference from primer-dependent artifacts[J]. Frontiers in Plant Science, 2022, 13:856109.doi:10.3389/fpls.2022.856109.
[7]	Maehara N, Kanzaki N, Aikawa T, Nakamura K. Potential vector switching in the evolution of Bursaphelenchus xylophilus group nematodes(Nematoda:Aphelenchoididae)[J]. Ecology and Evolution, 2020, 10(24):14320-14329.doi:10.1002/ece3.7033.
[8]	Maehara N, Aikawa T, Kanzaki N, Nakamura K. Transfer of four isolates of Bursaphelenchus doui into Monochamus alternatus and potential vector switching of the nematode[J]. Nematology, 2022, 24(8):855-861.doi:10.1163/15685411-bja10173.
[9]	Aikawa T, Ozawa S, Maehara N, Masuya H, Nakamura K, Kanzaki N. Discovery of a phoretic association between Bursaphelenchus doui(Nematoda:Aphelenchoididae)and Monochamus saltuarius and Acalolepta sejuncta(Coleoptera:Cerambycidae)[J]. Nematology, 2019, 22(6):713-722.doi:10.1163/15685411-00003334.
[10]	周沁莹, 朱曼, 黄辉, 黄晓玮. 植物寄生线虫的化学感受系统研究进展[J]. 中国科学:生命科学, 2019, 49(7):828-838.doi:10.1360/SSV-2019-0092.
	Zhou Q Y, Zhu M, Huang H, Huang X W. Chemosensory system of plant parasitic nematodes[J]. Scientia Sinica(Vitae), 2019, 49(7):828-838.
[11]	You J, Pan F J, Wang S, Wang Y, Hu Y F. FMRFamide-like peptide 22 influences the head movement,host finding,and infection of Heterodera glycines[J]. Frontiers in Plant Science, 2021, 12:673354.doi:10.3389/fpls.2021.673354.
[12]	尤佳. 大豆孢囊线虫早期预寄生相关基因鉴定及FMRF酰胺多肽(FLPs)基因克隆和功能初探[D]. 长春: 中国科学院东北地理与农业生态研究所, 2019.
	You J. Identification of the parasitism-related genes,cloning and preliminary functional analysis of the FMRFamide neuropeptide gene from the soybean cyst nematode Heterodera glycines in the early stage[D]. Changchun: Northeast Institute of Geography and Agroecology,Chinese Academy of Sciences, 2019.
[13]	Kamaraju D, Chatterjee M, Papolu P K, Shivakumara T N, Sreevathsa R, Hada A, Rao U. Host-induced RNA interference targeting the neuromotor gene FMRFamide-like peptide-14(Mi-flp14)perturbs Meloidogyne incognita parasitic success in eggplant[J]. Plant Cell Reports, 2024, 43(7):178.doi:10.1007/s00299-024-03259-y.
[14]	Banakar P, Hada A, Papolu P K, Rao U. Simultaneous RNAi knockdown of three FMRFamide-like peptide genes,Mi-flp1,Mi-flp12,and Mi-flp18 provides resistance to root-knot nematode,Meloidogyne incognita[J]. Frontiers in Microbiology, 2020, 11:573916.doi:10.3389/fmicb.2020.573916.
[15]	Kumari C, Dutta T K, Chaudhary S, Banakar P, Papolu P K, Rao U. Molecular characterization of FMRFamide-like peptides in Meloidogyne graminicola and analysis of their knockdown effect on nematode infectivity[J]. Gene, 2017, 619:50-60.doi:10.1016/j.gene.2017.03.042
[16]	Hada A, Kumari C, Phani V, Singh D, Chinnusamy V, Rao U. Host-induced silencing of FMRFamide-like peptide genes,flp-1 and flp-12,in rice impairs reproductive fitness of the root-knot nematode Meloidogyne graminicola[J]. Frontiers in Plant Science, 2020, 11:1.doi:10.3389/fpls.2020.00894.
[17]	Atkinson H J, Isaac R E, Harris P D, Sharpe C M. FMRFamide like immunoreactivity within the nervous system of the nematodes Panagrellus redivius,Caenorhabditis elegans and Heterodera glycines[J]. Journal of Zoology, 1988, 216(4):663-671.doi:10.1111/j.1469-7998.1988.tb02464.x.
[18]	肖罗. 松材线虫(Bursaphelenchus xylophilus)flp基因与香蕉穿孔线虫(Radopholus similes)Rs-eng-1基因的克隆和功能分析[D]. 长沙: 湖南农业大学, 2008.
	Xiao L. Cloning and functional analysis of the flp genes in Bursaphelenchus xylophilus and the Rs-eng-1 gene in Radopholus similes[D]. Changsha: Hunan Agricultural University, 2008.
[19]	Huang Q X, Cheng X Y, Mao Z C. Wang Y S, Zhao L L, et al. MicroRNA discovery and analysis of pinewood nematode Bursaphelenchus xylophilus by deep sequencing[J]. PLoS One, 2010, 5(10):e13271.doi:10.1371/journal.pone.0013271.
[20]	林世锋, 郭立, 简恒, 张国珍. 松材线虫体前端特异cDNA文库的构建及细胞壁松弛蛋白基因的克隆与初步分析[J]. 植物病理学报, 2008, 38(3):283-291.doi:10.13926/j.cnki.apps.2008.03.018.
	Lin S F, Guo L, Jian H, Zhang G Z. Construction of specific cDNA library and cloning of expansin gene from the anterior end of Bursaphelenchus xylophilus[J]. Acta Phytopathologica Sinica, 2008, 38(3):283-291.
[21]	Li C, Kim K. Family of FLP peptides in Caenorhabditis elegans and related nematodes[J]. Frontiers in Endocrinology, 2014, 5:150.doi:10.3389/fendo.2014.00150.
[22]	Dayi M. Evolution of parasitism genes in the plant parasitic nematodes[J]. Scientific Reports, 2024, 14(1):3733.doi:10.1038/s41598-024-54330-3.
[23]	McVeigh P, Leech S, Mair G R, Marks N J, Geary T G, Maule A G. Analysis of FMRFamide-like peptide(FLP)diversity in Phylum nematoda[J]. International Journal for Parasitology, 2005, 35(10):1043-1060.doi:10.1016/j.ijpara.2005.05.010. pmid: 16076468
[24]	Rao U, Thakur P K, Prasad G N, Banakar P, Kumar M. Identification of neuropeptides,flp-1 and flp-12 targeting neuromuscular system of rice root knot nematode(RRKN)Meloidogyne graminicola[J]. Bioinformation, 2013, 9(4):182-186.doi:10.6026/97320630009182.
[25]	柳建发, 王中全. 医学寄生虫学[M]. 北京: 中国医药科技出版社, 2017.
	Liu J F, Wang Z Q. Medical parasitology[M]. Beijing: China Medical Science Press, 2017.
[26]	王天阳. 南方根结线虫flp-16、flp-27、flp-31基因的克隆及其表达原位杂交分析[D]. 北京: 中国农业科学院, 2008.doi:10.7666/d.Y1422213.
	Wang T Y. Cloning and expressed localization using in situ hybridization of FMRFamide-like peptides encoding genes Mi-flp-16/27/31 of Meloidogyne incognita[D]. Beijing: Chinese Academy of Agricultural Sciences, 2008.
[27]	芮凯. 海南水稻根结线虫动态、品种抗性及入侵机理研究[D]. 兰州: 甘肃农业大学, 2019.doi:10.27025/d.cnki.ggsnu.2019.000262.
	Rui K. The research on the dynamics and variety resistance and invasion mechanism of Meloidogyne graminicola in Hainan[D]. Lanzhou: Gansu Agricultural University, 2019.
[28]	彭焕, 胡先奇, 黄文坤, 贺文婷, 彭德良. 马铃薯腐烂茎线虫类FMRF酰胺多肽基因(Dd-flp-1)的克隆与表达定位分析[J]. 农业生物技术学报, 2011, 19(5):924-931.doi:10.3969/j.issn.1674-7968.2011.05.020.
	Peng H, Hu X Q, Huang W K, He W T, Peng D L. Cloning and localization analysis of a novel FMRFamide-like neuropeptide gene(Dd-flp-1)from migration plant-parasitic nematode(Ditylenchus destructor)on sweet-potato in China[J]. Journal of Agricultural Biotechnology, 2011, 19(05):924-931.

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