昆虫学报 ›› 2020, Vol. 63 ›› Issue (10): 1159-1170.doi: 10.16380/j.kcxb.2020.10.001

• 研究论文 •    下一篇

花椒窄吉丁触角转录组及嗅觉相关基因分析

巩雪芳, 谢寿安*, 杨平, 郭莉, 陈迪, 车显荣, 王延来, 吕淑杰   

  1. (西北农林科技大学林学院, 陕西杨陵 712100)
  • 出版日期:2020-10-20 发布日期:2020-11-06

Analysis of the antennal transcriptome and olfaction-related genes of Agrilus zanthoxylumi (Coleoptera: Buprestidae)

GONG Xue-Fang, XIE Shou-An*, YANG Ping, GUO Li, CHEN Di, CHE Xian-Rong, WANG Yan-Lai, LÜ Shu-Jie   

  1.  (College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100, China)
  • Online:2020-10-20 Published:2020-11-06

摘要: 【目的】建立花椒窄吉丁Agrilus zanthoxylumi成虫触角转录组数据库,挖掘嗅觉相关基因,为今后研究其触角的化学感受机制及生物防控提供理论支撑。【方法】采用高通量测序平台Illumina NovaSeq 6000对花椒窄吉丁雌雄成虫触角进行转录组测序,用Trinity软件对获得的高质量reads进行序列拼接与组装;使用BLAST软件将触角转录组数据比对NR, NT, Swiss-Prot, GO, KEGG, BLASTX, eggNOG, Pfam, TmHMM, SignalP, KO, Map, BLASTP和RNAMMER公共数据库;基于初步筛选到的花椒窄吉丁候选气味结合蛋白(odorant binding protein, OBP)和化学感受蛋白(chemosensory protein, CSP)以及其他鞘翅目昆虫的同源蛋白的核苷酸序列,利用MEGA软件进行系统进化分析。运用RPKM (reads per kilobase per million mapped reads)值对嗅觉相关基因表达量进行分析。【结果】花椒窄吉丁雌雄成虫触角转录组测序共获得36 209条基因和90 982条转录本,其N50分别为2 103和2 523 bp,组装完整性较高。注释到NR数据库的基因最多(41.62%),其中与赤拟谷盗Tribolium castaneum相似基因所占比例最高(19%)。在GO数据库中比对到11 614个基因,按功能分为细胞组分、分子功能与生物学进程三大类57个分支,其中分子功能大类中的结合(70.57%)与催化活性(45.51%)相关基因占比最多;KEGG代谢途径分析表明,7 427条基因参与了5类代谢通路,其中涉及信号转导的基因最多,为815条;筛选到7个候选OBP基因和5个具有全长开放阅读框的CSP基因,其编码蛋白均具有化学感受蛋白家族的典型特征。系统进化分析表明,花椒窄吉丁OBPs和CSPs分别与苹果小吉丁A. mali的OBPs和CSPs氨基酸序列一致性最高。RPKM值表明,嗅觉相关基因AzanOBP1和AzanOBP2在雌成虫触角中不表达,在雄成虫触角中微量表达;AzanOBP3在雄成虫触角中高丰度表达。【结论】首次获得了花椒窄吉丁成虫触角转录组数据,筛选到了花椒窄吉丁OBP, CSP, Or, IR和SNMP等的嗅觉相关基因。推测触角中高丰度表达的OBPs对雄成虫识别同类异性释放的信息素或寄主植物释放的挥发物起关键作用。研究结果可为花椒窄吉丁化学感受基因功能分析及嗅觉感受机制研究奠定分子基础。

关键词: 花椒窄吉丁, 触角转录组, 基因注释, 气味结合蛋白, 化学感受蛋白

Abstract: 【Aim】 To establish the antennal transcriptome database of Agrilus zanthoxylumi adult and to explore target genes related to olfaction, so as to provide the theoretical basis for the future study of the chemosensory mechanism of its antenna and its biological control. 【Methods】 High-throughput sequencing platform Illumina NovaSeq 6000 was used for transcriptome sequencing of the antennae of female and male adults of A. zanthoxylumiTrinity software was used for sequence splicing and assembly of the high-quality reads obtained. BLAST software was used to compare the antennal transcriptome data with public databases NR, NT, Swiss-Prot, GO, KEGG, BLASTX, eggNOG, Pfam, TmHMM, SignalP, KO, Map, BLASTP and RNAMMER. Phylogenetic analysis was carried out using MEGA software based on the nucleotide sequences of the preliminarily screened candidate odorant binding proteins (OBPs) and chemosensory proteins (CSPs) of A.zanthoxylumi, and homologous proteins from other coleopteran insects. The RPKM (reads per kilobase per million mapped reads) value was used to evaluate the expression levels of olfaction-related genes. 【Results】 By sequencing of the antennal transcriptome of female and male adults of A. zanthoxylumi, a total of 36 209 unigenes and 90 982 transcripts were obtained with the N50 of 2 103 and 2 523 bp, respectively, indicating the high completeness of assembly. Most unigenes (41.62%) were annotated to the NR database, with the highest proportion (19%) of genes similar to those of Tribolium castaneum. In the GO database, 11 614 unigenes were annotated and divided into 57 branches of three functional categories, cellular component, molecular function and biological process, among which binding (70.57%) and catalytic activity (45.51%) related unigenes in the category of molecular function were the most. The KEGG metabolic pathway analysis indicated that 7 427 unigenes are involved in five categories of metabolic pathways, with the most unigenes (815) involved in signal transduction. Seven candidate OBP genes and 5 CSP genes with full-length open reading frame were screened, and their encoded proteins have the typical characteristics of chemosensory protein family. The phylogenetic analysis showed that the OBPs and CSPs of A. zanthoxylumi have the highest sequence identity with the OBPs and CSPs of A. mali, respectively. The RPKM values showed that AzanOBP1 and AzanOBP2 were not expressed in the antennae of female adults, but slightly expressed in the antennae of male adults, and AzanOBP3 was expressed with high abundance in the antennae of male adults. 【Conclusion】 For the first time, the antennal transcriptome data of A. zanthoxylumi were obtained, and the olfaction-related OBP, CSP, OR, IR and SNMP genes of A. zanthoxylumi were screened. It is speculated that OBPs expressed with high abundance in the antennae play a key role in identifying pheromones released by female adults of the same species or the volatiles released by host plants. The results can lay a molecular basis for further studying the functions of chemosensory genes and olfactory sensitivity mechanism of A. zanthoxylumi.

Key words: Agrilus zanthoxylumi, antennal transcriptome, gene annotation, odorant binding protein, chemosensory protein