豆伞滑刃线虫类FMRF酰胺多肽基因的克隆与分析

doi:10.7668/hbnxb.20195409

摘要/Abstract

摘要：

为探究豆伞滑刃线虫与寄主植物互作的分子机制,通过构建豆伞滑刃线虫体前端特异cDNA文库与RACE技术相结合的方法克隆获得1个豆伞滑刃线虫类FMRF酰胺多肽基因Bd-FLP-12,并应用生物信息学方法进行序列分析,应用Southern杂交和半定量RT-PCR方法分别检测基因拷贝数和在不同发育阶段的表达水平。序列分析表明,Bd-FLP-12编码蛋白由90个氨基酸残基组成,其中含有1个信号肽序列和1个FLP-12成熟肽序列,无跨膜结构域,属于分泌型蛋白。根据Southern杂交结果推测,Bd-FLP-12基因在豆伞滑刃线虫基因组中为单拷贝。半定量RT-PCR结果显示,Bd-FLP-12在豆伞滑刃线虫各龄期均有表达,但在卵期相对比较微弱。综上,首次在豆伞滑刃线虫中克隆获得Bd-FLP-12基因全长序列,解析了该基因的结构、性质和表达特征,为进一步研究该基因的功能奠定了基础。

关键词: 豆伞滑刃线虫, 类FMRF酰胺多肽, 生物信息学, Southern杂交, 表达分析

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

中图分类号:

S432.45
S763

林世锋, 王仁刚, 王自力, 李莉, 陈越男, 吴沙沙, 刘国琴. 豆伞滑刃线虫类FMRF酰胺多肽基因的克隆与分析[J]. 华北农学报, 2025, 40(3): 166-172. doi: 10.7668/hbnxb.20195409.

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.

http://www.hbnxb.net/CN/Y2025/V40/I3/166

图/表 8

图1 以消减杂交产物为模板扩增hsp90基因的凝胶电泳结果 M.DL2000 Marker;1.未经消减杂交cDNA为模板;2.第一轮消减杂交cDNA为模板;3.第二轮消减杂交cDNA为模板。

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.

图2 豆伞滑刃线虫前后端cDNA反向Northern杂交 A.与体前端cDNA杂交;B.与体后端cDNA杂交。单克隆菌落的编号以字母和数字的组合表示,字母代表列,数字代表行。

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.

图3 编号Bd-m36基因5'RACE扩增结果 M.DL2000 Marker;1.第一轮PCR产物;2.第二轮PCR产物。

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

图4 克隆获得Bd-FLP-12基因的全长cDNA序列(A)及其基因组DNA序列(B) A.小写部分为非编码区;大写部分为编码区及编码的氨基酸序列;*为终止密码子;方框内为信号肽;黑色阴影部分为预测的RNKFEFIRF酰胺成熟肽序列;下划线部分为poly(A)加尾信号(AATAAA)。B.方框部分为外显子,横线部分为内含子;GT是内含子的初始碱基,AG是内含子的结束碱基;数字是对应部分的序列长度。

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.

图5 Bd-FLP-12基因组杂交分析 M.DNA在琼脂糖凝胶中迁移的标准大小(kb);N.豆伞滑刃线虫总DNA;F.灰葡萄孢菌总DNA;1.BamHⅠ酶切基因组DNA;2.Hind Ⅲ酶切基因组DNA。

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.

图6 豆伞滑刃线虫Bd-FLP-12蛋白信号肽(A)和跨膜结构域(B)预测

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

图7 豆伞滑刃线虫Bd-FLP-12蛋白与其他植物寄生线虫同源蛋白的氨基酸序列比对 1.豆伞滑刃线虫(Bd-FLP-12);2.松材线虫(ABR09285);3.水稻干尖线虫(KAI6187848);4.鼻状杯环线虫(CAJ0609964);5.斯氏线虫(KAK0396706);6.有齿食道口线虫(KHJ99768);7.小根结线虫(ABB49052);8.禾谷孢囊线虫(AEN71840);9.南方根结线虫(AAX19364);10.马铃薯白线虫(CAC32452);RNKFEFIRF.FLP-12典型氨基酸序列。

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.

图8 用半定量RT-PCR分析Bd-FLP-12基因的发育表达模式 1.卵;2.二龄幼虫;3.三龄幼虫;4.四龄幼虫;5.雌性成虫;6.雄性成虫。

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.

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