花椒窄吉丁转录组及化学感受相关基因的分析
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摘要
【目的】建立花椒窄吉丁Agrilus zanthoxylumi转录组数据库,挖掘其基因组数据。【方法】采用高通量测序平台Illumina NovaSeq6000对花椒窄吉丁成虫不同组织(触角、头、胸、腹、足、翅)进行转录组测序、序列组装,使用BLAST软件将花椒窄吉丁unigenes序列与公共数据库进行比对,BLAST同源性搜索的方法从中筛选出与其化学感受相关的基因,并与其他已研究发表的鞘翅目昆虫化学感受相关基因的核酸序列比对,进行系统发育分析。利用RPKM值对这些基因在花椒窄吉丁成虫不同组织中的表达量进行分析。【结果】经测序及序列拼接后共得到80 320条unigenes和169 398条contigs,其G+C比例分别是37.63%和39.18%,平均长度分别为828.75和1 084.33 bp; unigenes长度主要分布在200～400 bp的共有37 374条,占全部unigenes的46.53%。在NR数据库中注释的与花椒窄吉丁转录组相似基因所属物种分布为赤拟谷盗Tribolium castaneum所占比例26.80%,其后依次是小家鼠Mus musculus(13.87%),蛀犀金龟Oryctes borbonicus(5.26%),山松甲虫Dendroctonus ponderosae(4.10%),黑蚁Lasius niger(2.36%),其他物种所占比例是47.61%。在GO数据库中比对到27 488个unigenes,可分为分子功能、细胞组分和生物学进程三大类共59个过程;利用KOG数据库功能注释分为25类,注释到unigenes最多功能类别的是普通功能,共4 666个,最少的是与细胞移动相关,共40个;将80 320个unigenes映射到KEGG数据库中,21 104条unigens注释到代谢通路35个,占26.27%,注释最多的代谢途径是转录途径,共涉及到2 037条unigenes。从NR数据库中共注释到8个气味受体(odorant receptor, OR)基因、7个离子型受体(ionotropic receptor, IR)基因和6个味觉受体(gustatory receptor, GR)基因,各组织中的基因表达量分析发现,这3类化学感受相关基因在成虫触角中的表达量显著高于其他组织中的表达量,雌、雄触角中表达量差异最显著的为AzanOR1,而在雌、雄成虫足中这3类化学感受相关基因表达量没有差异。【结论】本研究首次获得了花椒窄吉丁转录组数据,为进一步研究花椒窄吉丁的基因功能奠定了分子基础。
【Aim】 To establish the transcriptome database of the Agrilus zanthoxylumi, and to obtain its genomic data. 【Methods】 The transcriptome of the different tissues(antennae, head, thorax, abdomen, leg, wing) of A. zanthoxylumi adults was sequenced using an Illumina NovaSeq 6000 platform, and the unigene sequences were compared with the homologous sequences in public databases by BLAST. Chemoreception-related genes were screened out by BLAST homology search, and their nucleotide sequences were compared with those of other reported chemoreception-related genes of Coleoptera insects for phylogenetic analysis. The expression levels of these genes in different tissues of A.zanthoxylumi adults were analyzed based on the RPKM value. 【Results】 After sequencing and sequence splicing, a total of 80 320 genes and 169 398 contigs were obtained. The G+C proportions of contigs and unigenes were 37.63% and 39.18%, respectively, and the average length of contigs and the average length of unigenes were 1 084.33 and 828.75 bp, respectively. There were 37 374 unigenes with a length distribution mainly in 200-400 bp, accounting for 46.53% of the total unigenes. Origin species distribution of similar genes with unigenes of A. zanthoxylumi annotated in the NR database was Tribolium castaneum(26.80%), Mus musculus(13.87%), Oryctes borbonicus(5.26%), Dendroctonus ponderosae(4.10%), Lasius niger(2.36%), and other species(47.61%). There were 27 488 unigenes aligned to the GO database, which can be divided into three functional categories including molecular function, cellular component and biological process in a total of 59 processes. According to the gene annotation against the KOG database, these unigenes can be divided into 25 categories. The most annotated functional category was the common function(4 666 unigenes), while the least annotated was cell movement-related(only 40 unigenes). When 80 320 unigenes were mapped to the KEGG database, 21 104 unigenes were grouped into 35 metabolic pathways, accounting for 26.27% of the total. The most annotated metabolic pathway was the transcription pathway(2 037 unigenes). By gene annotation against the NR database, we found eight odorant receptor(OR) genes, seven ionotropic receptor(IR) genes and six gustatory receptor(GR) genes. Tissue expression profiles showed that the three kinds of chemoreception-related genes(OR, IR and GR genes) had significantly higher expression levels in the antennae than in other tissues of adults, and the expression level of AzanOR1 in the antennae showed the most significant difference between female and male, while the expression levels of the three kinds of chemoreception-related genes in the legs of adults showed no difference between female and male. 【Conclusion】 This study obtained the transcriptome data of A. zanthoxylumi. The results provide a molecular foundation for further studying the gene function in A. zanthoxylumi.
【Aim】 To establish the transcriptome database of the Agrilus zanthoxylumi, and to obtain its genomic data. 【Methods】 The transcriptome of the different tissues(antennae, head, thorax, abdomen, leg, wing) of A. zanthoxylumi adults was sequenced using an Illumina NovaSeq 6000 platform, and the unigene sequences were compared with the homologous sequences in public databases by BLAST. Chemoreception-related genes were screened out by BLAST homology search, and their nucleotide sequences were compared with those of other reported chemoreception-related genes of Coleoptera insects for phylogenetic analysis. The expression levels of these genes in different tissues of A.zanthoxylumi adults were analyzed based on the RPKM value. 【Results】 After sequencing and sequence splicing, a total of 80 320 genes and 169 398 contigs were obtained. The G+C proportions of contigs and unigenes were 37.63% and 39.18%, respectively, and the average length of contigs and the average length of unigenes were 1 084.33 and 828.75 bp, respectively. There were 37 374 unigenes with a length distribution mainly in 200-400 bp, accounting for 46.53% of the total unigenes. Origin species distribution of similar genes with unigenes of A. zanthoxylumi annotated in the NR database was Tribolium castaneum(26.80%), Mus musculus(13.87%), Oryctes borbonicus(5.26%), Dendroctonus ponderosae(4.10%), Lasius niger(2.36%), and other species(47.61%). There were 27 488 unigenes aligned to the GO database, which can be divided into three functional categories including molecular function, cellular component and biological process in a total of 59 processes. According to the gene annotation against the KOG database, these unigenes can be divided into 25 categories. The most annotated functional category was the common function(4 666 unigenes), while the least annotated was cell movement-related(only 40 unigenes). When 80 320 unigenes were mapped to the KEGG database, 21 104 unigenes were grouped into 35 metabolic pathways, accounting for 26.27% of the total. The most annotated metabolic pathway was the transcription pathway(2 037 unigenes). By gene annotation against the NR database, we found eight odorant receptor(OR) genes, seven ionotropic receptor(IR) genes and six gustatory receptor(GR) genes. Tissue expression profiles showed that the three kinds of chemoreception-related genes(OR, IR and GR genes) had significantly higher expression levels in the antennae than in other tissues of adults, and the expression level of AzanOR1 in the antennae showed the most significant difference between female and male, while the expression levels of the three kinds of chemoreception-related genes in the legs of adults showed no difference between female and male. 【Conclusion】 This study obtained the transcriptome data of A. zanthoxylumi. The results provide a molecular foundation for further studying the gene function in A. zanthoxylumi.
引文
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Chen XX,Ren SX,Zhang F,Cai WZ,Zeng FR,Zhang WQ,2013.Pest control mechanism and sustainable utilization.Chin.J.Appl.Entomol.,50(1):9-18.[陈学新,任顺祥,张帆,彩万志,曾凡荣,张文庆,2013.天敌昆虫控害机制与可持续利用.应用昆虫学报,50(1):9-18]
Clyne PJ,Warr CG,Freeman MR,Lessing D,Kim J,Carlson JR,1999.A novel family of divergent seven-transmembrane proteins:candidate odorant receptors in Drosophila.Neuron,22:327-338.
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Cui YY,1990.Rules of leaf occurrence and prevention of Zanthoxylum bungeanum.Hebei Fruits,19(2):36-37.[崔艳鱼,1990.花椒潜叶发生规律及防治.河北果树,19(2):36-37]
Gayral P,Weinert L,Chiari Y,Tsagkogeorga G,Ballenghien M,Galtier N,2011.Next-generation sequencing of transcriptomes:a guide to RNA isolation in nonmodel animals.Mol.Ecol.Res.,11(4):650-661.
Grabherr MG,Haas BJ,Yassour M,Levin JZ,Thompson DA,Amit I,Adiconis X,Fan L,Raychowdhury R,Zeng QD,Chen Z,Mauceli E,Hacohen N,Gnirke A,Rhind N,di Palma F,Birren BW,Nusbaum C,Lindblad-Toh K,Friedman N,Regev A,2011.Fulllength transcriptome assembly from RNA-Seq data without a reference genome.Nat.Biotechnol.,9(7):644-652.
Hansen KD,Irizarry RA,Wu ZJ,2012.Removing technical variability in RNA-seq data using conditional quantile normalization.Biostatistics,13(2):204-216.
He HL,Bin SY,Wu ZZ,Lin JT,2012.Transcriptome characteristics of Phyllotreta striolata(Fabricius)(Coleoptera:Chrysomelidae)analyzed by using Illumina’s Solexa sequencing technology.Acta Entomol.Sin.,55(1):1-11.[贺华良,宾淑英,吴仲真,林进添,2012.基于Solexa高通量测序的黄曲条跳甲转录组学研究.昆虫学报,55(1):1-11]
He K,1991.Wound fluid glue and treatment of Zanthoxylum bungeanum.Gansu Agric.Sci.Technol.,(1):31.[何凱,1991.花椒树的伤口流胶及治疗.甘肃农业科技,(1):31]
Kang KG,Wang YP,Qiang L,Shi HQ,Chen H,2007.A study on chemical control test of Agrilus zanthoxylumi Hou and Feng.J.Northwest For.Univ.,22(6):105-107.[康克功,王永平,强磊,石和芹,陈辉,2007.花椒窄吉丁化学防治研究.西北林学院学报,22(6):105-107]
Kwon JY,Dahanukar A,Weiss LA,Carlson JR,2007.The molecular basis of CO2reception in Drosophila.Proc.Natl.Acad.Sci.USA,104(9):3574-3578.
Liu HL,Zheng LM,Liu QQ,Quan FS,Zhang Y,2013.Studies on the transcriptomes of non-model organisms.Hereditas(Beijing),35(8):955-970.[刘红亮,郑丽明,刘青青,权富生,张涌,2013.非模式生物转录组研究.遗传,35(8):955-970]
Nguyen CT,Lim S,Lee JG,Lee EJ,2017.Vc BBX,Vc MYB21 and VcR2R3MYB transcription factors are involved in UV-B-induced anthocyanin biosynthesis in the peel of harvested blueberry fruit.J.Agric.Food Chem.,65(10):2066-2073.
Robertson HM,Kent LB,2009.Evolution of the gene lineage encoding the carbon dioxide receptor in insects.J.Insect Sci.,9:19.
Robertson HM,Warr CG,Carlson JR,2003.Molecular evolution of the insect chemoreceptor gene super family in Drosophila melanogaster.Proc.Natl.Acad.Sci.USA,100(s2):14537-14542.
Sangwan RS,Tripathi S,Singh J,Narnoliya LK,Sangwan NS,2013.De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural,functional and regulatory genes with special reference to secondary metabolism.Gene,525(2):58-76.
Sun YZ,Li ZF,1988.A preliminary study of Agrilus zanthoxylumi.Plant Prot.,14(2):23-24.[孙益知,李忠锋,1988.花椒吉丁虫的初步研究.植物保护,14(2):23-24]
Vosshall LB,Amrein H,Morozov PS,Rzhetsky A,Axel R,1999.Aspatial map of olfactory receptor expression in the Drosophila antenna.Cell,96:725-736.
Wang WL,Feng CC,Hong M,Yue JW,Huhebateer,Liu CX,2017.The comparative transcriptome analysis of Parabronema skrjabini at different developmental stages.Sci.Agric.Sin.,50(23):4644-4655.[王文龙,冯陈晨,红梅,岳建伟,呼和巴特尔,刘春霞,2017.不同发育阶段斯氏副柔线虫比较转录组学分析.中国农业科学,50(23):4644-4655]
Wang XC,Tan HL,Chen Z,Meng LZ,Wang WB,Fan SC,2015.Assembly and characterization of the transcriptome and development of SSR markers in Forsythia suspensa based on RNA-Seq technology.Sci.Sin.Vit.,45(3):301-310.[王兴春,谭河林,陈钊,孟令芝,王文斌,范圣此,2015.基于RNA-Seq技术的连翘转录组组装与分析及SSR分子标记的开发.中国科学:生命科学,45(3):301-310]
Wu H,2006.Biological characteristics and control of Agrilus zanthoxylum.Chin.Bull.Entomol.,43(2):236-239.[吴海,2006.花椒窄吉丁的生物学特性及防治.昆虫知识,43(2):236-239]
Xu L,Yang P,Yuan S,Feng Y,Xu H,Cao Y,Ming J,2016.Transcriptome analysis identifies key candidate genes mediating purple ovary coloration in Asiatic hybrid lilies.Int.J.Mol.Sci.,17(11):1881-1888.
Yang F,Huang LH,Zhang AB,2014.High-throughput transcriptome sequencing technology and its applications in Lepidoptera.Acta Entomol.Sin.,57(8):991-1000.[杨帆,黄立华,张爱兵,2014.高通量转录组测序技术及其在鳞翅目昆虫上的应用.昆虫学报,57(8):991-1000]
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