摘要
【目的】本研究旨在鉴定云杉花墨天牛Monochamus saltuarius化学感受蛋白(chemosensory protein,CSP)基因,分析其时空表达谱,并阐明触角特异性高表达的CSPs与寄主油松Pinus tabulaeformis主要挥发性化合物的配体结合特性,为开发基于CSPs的绿色防控分子靶标提供依据。【方法】基于云杉花墨天牛转录组数据鉴定CSP基因,并利用生物信息学方法分析其表达谱;采用邻接法对云杉花墨天牛与其他鞘翅目(Coleoptera)昆虫的CSP基于氨基酸序列进行系统发育分析。根据已报道的云杉花墨天牛转录组数据中的TPM(tags per million)值,分析CSP基因在其取食不同植物(阔叶红松Pinus koraiensis和油松)的4龄幼虫中及成虫不同性别个体中的表达丰度,并使用qRT-PCR检测云杉花墨天牛CSP基因在成虫不同组织(触角、头、胸、腹、翅和足)中的表达量;通过原核表达和荧光竞争结合实验测定MsalCSP1重组蛋白与6种油松主要挥发性化合物(D-柠檬烯、月桂烯、3-蒈烯、α-蒎烯、β-蒎烯和莰烯)的结合特征。【结果】从云杉花墨天牛转录组数据中共鉴定出16个CSP基因(MsalCSP 1-16);系统发育分析显示12个CSPs与松墨天牛Monochamus alternatus的CSPs聚集为10个同源类群,表明进化关系相近。转录组分析显示,6个CSP基因在幼虫和成虫期均表达,在取食不同寄主植物的4龄幼虫中及成虫不同性别之间6个CSP基因表达量也存在差异;qRT-PCR结果显示,MsalCSP 1和MsalCSP 5在成虫触角中特异性高表达,MsalCSP 13和MsalCSP 15在头部高表达,MsalCSP 11在翅中高表达,MsalCSP 3和MsalCSP 13在足中高表达。荧光竞争结合结果显示,MsalCSP1重组蛋白与油松6种主要挥发性化合物D-柠檬烯、月桂烯、3-蒈烯、α-蒎烯、β-蒎烯和莰烯的结合能力强,结合常数(binding constant,K i)值分别为0.87,0.70,0.91,0.85,1.09和0.70μmol/L。【结论】本研究首次鉴定出云杉花墨天牛的16个CSP基因,并明确其在4龄幼虫和成虫不同组织中的表达特征,推测成虫触角高表达的MsalCSP1在云杉花墨天牛定位寄主植物过程中发挥重要作用。
【Aim】This study aims to identify the chemosensory protein(CSP)genes,analyze their spatiotemporal expression profiles in Monochamus saltuarius,and clarify the ligand binding characteristics of antennae-enriched CSPs with the main volatile compounds of the host Pinus tabulaeformis,so as to provide a basis for developing CSP-based molecular targets for green prevention and control.【Methods】Based on the transcriptome data of M.saltuarius,the CSP genes and their expression profiling were identified and analyzed by bioinformatics methods.Phylogenetic analysis of CSPs from M.saltuarius and other coleopteran insects based on amino acid sequences was performed using the neighbor-joining method.The expression abundance of CSP genes in the 4th instar larvae fed on different host plants(Pinus koraiensis and P.tabulaeformis)and adults of different sexes of M.saltuarius were analyzed based on the TPM(tags per million)values of CSP genes in the reported transcriptomes of M.saltuarius.The expression levels of CSP genes in different tissues of adults(antenna,head,throax,abdomen,wing and leg)of M.saltuarius were detected by qRT-PCR.The binding characteristics of the recombinant MsalCSP1 with six major volatile compounds(D-limonene,myrcene,3-carene,α-pinene,β-pinene and camphene)of P.tabulaeformis were assayed by prokaryotic expression and fluorescence competitive binding assay.【Results】A total of 16 CSP genes(MsalCSP 1-16)were identified from the transcriptome data of M.saltuarius.Phylogenetic analysis showed that 12 CSPs were clustered into 10 homologous groups with the CSPs of Monochamus alternatus,indicating the similar evolutionary relationship.Transcriptome analysis showed that six CSP genes were expressed in both the larval and adult stages,and there were differences in the expression levels of the six CSP genes in the 4th instar larvae fed on different host plants and adults of different sexes.qRT-PCR results showed that MsalCSP 1 and MsalCSP 5 were specifically highly expressed in the adult antennae,MsalCSP 13 and MsalCSP 15 were highly expressed in the head,MsalCSP 11 was highly expressed in the wing,MsalCSP 3 and MsalCSP 13 were highly expressed in the leg.The fluorescence competitive binding assay result indicated that the recombinant MsalCSP1 had strong binding abilities with six major volatile compounds(D-limonene,myrcene,3-carene,α-pinene,β-pinene and camphene)of P.tabulaeformis,with the binding constant(K i)values of 0.87,0.70,0.91,0.85,1.09 and 0.70μmol/L,respectively.【Conclusion】A total of 16 CSP genes of M.saltuarius were first identified in this study,and their expression characteristics were also clarified across the 4th instar larvae and various adult tissues.It is speculated that MsalCSP1 highly expressed in the adult antennae plays an important role in locating host plants.
作者
张立民
陈伟
李真祥
王军
杨静
王恺
罗晨
张苏芳
宗世祥
孙江华
刘芳华
ZHANG Li-Min;CHEN Wei;LI Zhen-Xiang;WANG Jun;YANG Jing;WANG Kai;LUO Chen;ZHANG Sun-Fang;ZONG Shi-Xiang;SUN Jiang-Hua;LIU Fang-Hua(Mulanweichang State-owned Forest Farm of Hebei Province,Chengde 068450,China;Hebei Basic Science Center for Biotic Interactions,Institute of Life Science and Green Development,College of Life Science,Hebei University,Baoding 071002,China;Key Laboratory of Forest Protection of National Forestry and Grassland Administration,Ecology and Nature Conservation Institute,Chinese Academy of Forestry,Beijing 100091,China;Beijing Key Laboratory for Forest Pest Control,Beijing Forestry University,Beijing 100083,China)
出处
《昆虫学报》
北大核心
2025年第11期1499-1509,共11页
Acta Entomologica Sinica
基金
河北大学青年拔尖人才项目(050001-513300201004)
河北省优秀青年科研创新团队(QNTD202405)。
关键词
云杉花墨天牛
化学感受蛋白
时空表达谱
气味结合特性
化学感受
寄主定位
Monochamus saltuarius
chemosensory protein
spatiotemporal expression profiles
odor binding characteristics
chemical sensation
host location
