【摘要】 【背景】二化螟（Chilo suppressalis）是我国水稻上的重要害虫之一，味觉受体（gustatory receptor,GR）基因在昆虫取食、产卵等过程中发挥重要作用。【目的】基于组学数据鉴定并克隆二化螟GR基因序列，明确其在不同发育阶段和成虫不同组织中的表达特性，为后续深入研究二化螟GR基因的功能打下基础。【方法】结合二化螟转录组数据和其他昆虫的GR基因序列，通过多重序列比对，鉴定二化螟GR基因。利用RT-PCR方法克隆获得二化螟GR基因的完整开放阅读框（open reading frame,ORF）序列，运用生物信息学分析工具对二化螟GR基因编码的氨基酸序列进行分子生物学特征、结构域等分析；基于最大似然法构建二化螟GR基因蛋白序列与其他昆虫GR基因的系统进化树；利用实时荧光定量PCR（RT-qPCR）方法分析GR基因在二化螟不同发育阶段（1—6龄幼虫和雌、雄成虫）和成虫不同组织（雌、雄成虫的触角、头、翅、腹、足）中的表达模式。【结果】鉴定并克隆得到5个二化螟GR基因，分别命名为CsupGR1—5,ORF长度为1 122—1 428 bp，编码氨基酸序列长度为373—475 aa，其中CsupGR2、CsupGR4、CsupGR5具有7个跨膜结构域，CsupGR1、CsupGR3具有8个跨膜结构域。系统进化分析显示，CsupGR1、CsupGR2与黑腹果蝇DmelGR63a及小菜蛾PxylGR7、PxylGR8亲缘关系较近，属于CO₂受体家族；CsupGR3与黑腹果蝇DmelGR43a及家蚕BmorGR9、BmorGR10亲缘关系较近，属于果糖/肌醇受体家族；CsupGR4、CsupGR5与黑腹果蝇DmelGR64及家蚕BmorGR5—8亲缘关系较近，属于糖受体家族。发育期表达谱分析表明，二化螟5个GR基因在不同发育时期均有表达，其中CsupGR1、CsupGR4均在雄成虫中高表达，CsupGR2在1龄幼虫和雄成虫中表达量最高，CsupGR3在成虫中高表达，CsupGR5在4龄幼虫中表达量最高；组织表达谱分析表明，5个GR基因在雌、雄成虫的各组织均有表达，其中CsupGR1、CsupGR5在成虫头部表达量高，CsupGR2—4在成虫触角部位高表达。【结论】鉴定克隆的5个二化螟GR基因均具有昆虫味觉受体基因的典型特征，且在成虫触角或头部高表达，推测这5个基因可能与二化螟识别和适应寄主植物有关。更多还原

【Abstract】 【Background】The rice striped stem borer,Chilo suppressalis,is one of the important rice pests in China.The gustatory receptors （GRs） play important roles in insect feeding,oviposition,and so on.【Objective】Based on transcriptomic and genomic data,the gustatory receptor gene sequences of C.suppressalis were identified and cloned in this study.Then,their expression characteristics at different developmental stages and in different tissues of adults were clarified.These results will lay a foundation for further study on the function of gustatory receptor genes in C.suppressalis.【Method】Combined with the transcriptome data of C.suppressalis and the GR sequences of other insects,the GR genes in C.suppressalis were identified by multiple sequence alignment.The complete open reading frame （ORF） sequences of the GR genes in C.suppressalis were cloned by RT-PCR,and the molecular biological characteristics and domains of the amino acid sequences encoded by the GR genes in C.suppressalis were analyzed by bioinformatics analysis tools.The phylogenetic tree of GR gene protein sequences of C.suppressalis and other insect GR genes was constructed based on the maximum likelihood method.The expression patterns of GR genes at different developmental stages （1-6instar larvae,female and male adults） and in different adult tissues （antennae,head,wing,abdomen and leg） of C.suppressalis were determined by real-time quantitative PCR （RT-qPCR）.【Result】Five gustatory receptor genes were identified and named as CsupGR1-5.The complete ORFs of these GR genes were between 1 122 and 1 428 bp in length,encoding 373-475 amino acids.Among them,CsupGR2,CsupGR4,CsupGR5 had seven transmembrane domains,CsupGR1,CsupGR3 had eight transmembrane domains.Phylogenetic analysis showed that CsupGR1 and CsupGR2 were closely related to Dmel GR63a in Drosophila melanogaster,PxylGR7 and PxylGR8 in Plutella xylostella,belonging to the CO₂ receptor family.CsupGR3 was closely related to DmelGR43a in D.melanogaster,Bmor GR9 and Bmor GR10 in Bombyx mori,belonging to the fructose/inositol receptor family.CsupGR4 and CsupGR5 were closely related to DmelGR64 in D.melanogaster and Bmor GR5-8 in B.mori,belonging to the sugar receptor family.The expression profile analysis showed that the five GR genes were all expressed at different developmental stages in C.suppressalis.Among them,CsupGR1 and CsupGR4 were highly expressed in male adults,CsupGR2 had the highest expression level in the 1st instar larvae and male adults,CsupGR3 was highly expressed in adults,and CsupGR5 had the highest expression level in the 4th instar larvae.Tissue expression profile analysis showed that these five GR genes were expressed in all tissues of male and female adults.CsupGR1 and CsupGR5 were highly expressed in the head of adults,and CsupGR2-4 were highly expressed in the antennae of adults.【Conclusion】The five GR genes of C.suppressalis identified in this study have the typical characteristics of insect gustatory receptors and are highly expressed in the antennae or heads of adults.It is speculated that these five genes may be related to the recognition and adaptation of C.suppressalis to host plants.更多还原