拟松材线虫G蛋白α亚基Bm...-gpa-1表达特性及功能_马芮.pdf

第 58 卷 第 4 期2 0 2 2 年 4 月林业科学SCIENTIASILVAESINICAEVol.58,No.4Apr.,2 0 2 2doi:10.11707/j.1001-7488.20220410收稿日期:2021-11-03;修回日期:2022-12-20。基金项目:国家自然科学基金项目(31670652;31870633)。胡加付为通讯作者。拟松材线虫 G 蛋白 亚基 Bmu-gpa-1 表达特性及功能马芮刘文义余红仕周立峰胡加付(浙江农林大学杭州 311300)摘要:【目的】研究与交配行为相关的拟松材线虫 G 蛋白 亚基 Bmu-gpa-1 基因的表达特性及生物学功能,为拟松材线虫的种内交配繁殖分子机制研究提供依据。【方法】根据拟松材线虫基因组数据设计引物克隆Bmu-gpa-1 基因。对 Bmu-gpa-1 进行序列分析、系统发育分析和蛋白结构预测等生物信息学分析。利用实时荧光定量 PCR 技术和原位杂交技术探究 Bmu-gpa-1 基因在拟松材线虫各个龄期的表达水平和表达部位,明确其时空动态表达特性。利用 RNA 干扰技术探究该基因在拟松材线虫交配繁殖行为中的作用,并通过实时荧光定量 PCR 技术检测 RNA 干扰的效率。【结果】Bmu-gpa-1 基因全长为 1 780 bp,CDS 全长为 1 065 bp,编码 354 个氨基酸。进化分析表示该基因与秀丽隐杆线虫 gpa-1 基因隶属于同一 G 蛋白 亚基家族。荧光定量 PCR 结果及转录组结果均表明 Bmu-gpa-1 基因在拟松材线虫各个发育阶段均有表达,2 龄期和 3 龄期表达水平较高,胚胎期次之,4 龄期和成虫期表达水平较低且基因表达水平具有雌、雄虫差异性,即雄虫的基因表达水平显著高于雌虫。原位杂交结果表明,Bmu-gpa-1 基因在拟松材线虫体内广泛表达,胚胎发育初期呈散点状表达,胚胎发育后期过渡到全胚胎表达,2 龄期全身表达,3 龄期和 4 龄期进一步集中在肠道和性腺处表达,成虫期则出现雌、雄虫差异性表达,即在雌虫的阴户、雄虫的交合刺和尾部表达。交配试验结果显示,沉默 Bmu-gpa-1 基因后,拟松材线虫雄虫与正常雌虫的交配次数显著增加,交配时长显著缩短,繁殖能力显著下降,而雌虫被干扰后在交配繁殖行为上则无明显异常,推测该基因可以负向调控拟松材线虫雄虫的交配行为,但对雌虫影响较弱。【结论】拟松材线虫 Bmu-gpa-1 基因是 G 蛋白 亚基家族中的一员,基因表达水平及表达部位皆具有雌、雄虫差异性。沉默该基因可以改变雄虫的交配行为,雄虫表现出交配次数增加、交配时长减短和交配后雌虫产卵数减少等交配质量下降特征,但对雌虫影响较弱,这说明 Bmu-gpa-1 基因对拟松材线虫雄虫的交配繁殖行为具有明显的调控功能。关键词:拟松材线虫;Bmu-gpa-1;交配行为;繁殖能力;雌、雄虫差异性中图分类号:S763文献标识码:A文章编号:1001-7488(2022)04-0095-09Expression Characteristics and Functions of G Protein Alpha Subunit Bmu-gpa-1 in Bursaphelenchus mucronatusMa RuiLiu WenyiYu HongshiZhou LifengHu Jiafu(Zhejiang Agriculture and Forestry UniversityHangzhou 311300)Abstract:【Objective】As a member of the pine wood nematode disease system,Bursaphelenchus mucronatus has caused serious ecological and economic losses in China due to its efficient and frequent mating behavior.We therefore investigated Bmu-gpa-1 gene of G protein that is related to mating behaviour,and its expression characteristics and its role in mating behavior in B.mucronatus,which would provide a basis for the study of the molecular mechanisms for mating and reproduction in B.mucronatus.【Method】Bmu-gpa-1 gene was cloned by PCR based on the genomic data of B.mucronatus.The sequence alignment,phylogenetic analysis and protein structure prediction were conducted to clarify its evolutional relationship with other species.The RT-qPCR and in situ hybridization were used to analyze the gene expression level and expression location in different development stages to reveal its temporal and spatial dynamic expression characteristics.The gene Bmu-gpa-1 was silenced by RNAi to investigate the effects on mating behavior and 林业科学58 卷reproductive capability.In the meanwhile,RT-qPCR was used to check the efficiency of RNAi.【Result】The full-length of Bmu-gpa-1 gene is 1 780 bp,including 1 056 bp CDS,encoding 354 amino acids.Phylogenetic analysis indicated that the gene belonged to the same G protein alpha subunit family as gpa-1 in Caenorhabditis elegans.The results of RT-qPCR and transcriptomes showed that Bmu-gpa-1 was expressed in all developmental stages of B.mucronatus,with the expression level highest in the second and third juvenile stages,followed by embryonic stage,and the expression level in the fourth juvenile stage and the adult stage was the lowest.Meanwhile,we found the gene expression level has sexual dimorphism,showing a higher expression level in the males than that in the females.The result of mRNA in situ hybridization showed that Bmu-gpa-1 was expressed broadly in the nematodes.In the embryonic stage,expression location changed dramatically from sporadic dots to whole embryos.In the second juvenile stages,it was expressed in the whole bodies.In the third and fourth juvenile stages,it was mostly located in the intestinal tracts and gonads.In the adult stage,the expression location had sexual dimorphism,it was seen in the vulvas of females,whereas it was only observed in the spicules and tails in males.In the mating test,the males after silencing Bmu-gpa-1 had more frequent and faster mating behavior events than those of controls,but had fewer offspring.However,there were no significant changes in the mating and reproductive behavior of the RNAi-treated females.It is speculated that Bmu-gpa-1 gene can negatively regulate the mating behavior for males,but not for females.【Conclusion】Bmu-gpa-1 is a member of the G protein alpha subunit family.It has sexual dimorphism in the expression level and location.Our result indicate the males treated by RNAi have stronger capacity in signal transduction and massive invalid frequent mating behaviors,but not happened in females.It is suggested that Bmu-gpa-1 plays a negative role in the signal transduction of male nematodes and regulates their mating behaviors and fecundity in B.mucronatus.Key words:Bursaphelenchus mucronatus;Bmu-gpa-1;mating behavior;fecundity;sexual dimorphism拟松材线虫(Bursaphelenchus mucronatus)是一种 松 树 寄 生 线 虫,与 外 来 物 种 松 材 线 虫(B.xylophilus)同属于松材线虫病害系统,已在国内外造成严重的生态损失及经济损失(汤勤等,1989;张治 宇 等,2004;汤 坚 等,2008;Sultana et al.,2013)。过去对拟松材线虫病害没有引起足够的重视,但近年来越来越多的研究报告显示拟松材线虫也具有较高的致病性(陈凤毛等,2010;周立峰,2017;薛美静等,2019)。高效频繁的交配行为