沟眶象味觉受体基因EscrGR8对雌成虫繁殖力的影响  

Effects of the gustatory receptor gene EscrGR 8 on the fecundity of female adults of Eucryptorrhynchus scrobiculatus(Coleptera:Curculioniclae)

作　　者：郭晓丽 文超 温俊宝[1,2] 温晓健 GUO Xiao-Li;WEN Chao;WEN Jun-Bao;WEN Xiao-Jian(State Key Laboratory of Efficient Production of Forest Resources,Beijing Forestry University,Beijing 100083,China;Beijing Key Laboratory for Forest Pest Control,Beijing Forestry University,Beijing 100083,China;School of Grassland Science,Beijing Forestry University,Beijing 100083,China;Key Laboratory of Forest Protection of National Forestry and Grassland Administration,Ecology and Nature Conservation Institute,Chinese Academy of Forestry,Beijing 100091,China)

机构地区：[1]北京林业大学,林木资源高效生产全国重点实验室,北京100083 [2]北京林业大学,林木有害生物防治北京市重点实验室,北京100083 [3]北京林业大学草业与草原学院,北京100083 [4]中国林业科学研究院森林生态环境与自然保护研究所,国家林业和草原局森林保护学重点实验室,北京100091

出　　处：《昆虫学报》2024年第3期307-317,共11页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(32071774,32271888)。

摘　　要：【目的】分析和阐明沟眶象Eucryptorrhynchus scrobiculatus味觉受体基因EscrGR8的分子特性和功能,揭示其调节雌成虫繁殖力的作用。【方法】基于沟眶象触角转录组数据库,采用RACE克隆EscrGR8的cDNA全长序列;利用qRT-PCR检测EscrGR8在沟眶象不同发育阶段(卵、1-6龄幼虫、蛹、雌成虫和雄成虫)和雌雄成虫组织(去除触角和喙的头、触角、口器、中肠、前足、睾丸、卵巢、雄虫交配器和雌虫产卵器)中的表达量。显微注射雌成虫dsRNA后0,6,12,24,36,48和72 h时利用qRT-PCR检测RNAi后EscrGR8的表达量。检测显微注射dsEscrGR8后1-5 d和6-11 d时沟眶象雌成虫在不同土壤含水量(0~10%,11%~20%,21%~40%,41%~60%,61%~80%和81%~100%)条件下的产卵选择率、总产卵数量和所产卵的孵化率,研究抑制EscrGR8表达对雌成虫产卵选择性和繁殖力的影响。【结果】成功克隆了沟眶象EscrGR8(GenBank登录号:OR836580)的cDNA全长序列,开放阅读框(ORF)长1251 bp,编码了416个氨基酸,具有6个跨膜结构域。多序列比对和系统发育进化分析结果表明,EscrGR8与其他昆虫的GR的氨基酸序列同源性普遍较低,其中与棉铃象甲Anthonomus grandis的AgraGR64f氨基酸序列一致性为30.96%。qRT-PCR检测结果表明,EscrGR8在沟眶象不同发育阶段和成虫组织中的表达量具有显著差异,EscrGR8在雌成虫中的表达量最高,在卵中的表达量最低;EscrGR8在雌成虫卵巢中特异性高表达。显微注射dsEscrGR8不仅在一定时间内显著降低EscrGR8的表达量,并且对雌成虫的产卵选择性具有显著影响。显微注射dsEscrGR8后1-5 d时沟眶象雌成虫在含水量21%~40%土壤条件下的产卵选择率和所产卵的孵化率与显微注射dsGFP的对照组相比分别显著降低了24.66%和15.83%;在含水量81%~100%土壤条件下的产卵选择率与显微注射dsGFP的对照组相比显著增加了28.39%,雌成虫所产卵几乎不孵化。【结论】本研究证实了味觉受体基�【Aim】To analyze and elucidate the molecular characteristics and function of the gustatory receptor gene EscrGR 8 in Eucryptorrhynchus scrobiculatus,and to reveal its role in the regulation of the fecundity of female adult.【Methods】Based on the antennal transcriptome database of E.scrobiculatus,the cDNA full-length sequence of EscrGR 8 was cloned using RACE.The expression levels of EscrGR 8 in different developmental stages(egg,1st-6th instar larvae,pupa,female adult,and male adult),and female and male adult tissues(head without antennae and proboscis,antennae,mouthparts,midgut,forefoot,testicles,ovaries,male copulatory organ and female ovipositor)of E.scrobiculatus were detected by qRT-PCR.The expression levels of EscrGR 8 at 0,6,12,24,36,48 and 72 h after RNAi through microinjection of dsRNA into female adults were detected by qRT-PCR.The oviposition selection rates,total numbers of eggs laid and hatching rates of eggs laid by female adults under different soil moisture conditions(0-10%,11%-20%,21%-40%,41%-60%,61%-80%,and 81%-100%)at 1-5 d and 6-11 d after ds EscrGR 8 microinjection were determined,and the effects of inhibition of the EscrGR 8 expression on the oviposition preference and fecundity of female adults were studied.【Results】The full-length cDNA sequence of EscrGR 8(GenBank accession no.:OR836580)of E.scrobiculatus was successfully cloned with the open reading frame(ORF)of 1251 bp in length,encoding 416 amino acids.EscrGR8 has six transmembrane domains.Multiple sequence alignment and phylogenetic analysis results showed that EscrGR8 has a low homology to the GRs of other insects,and the amino acid sequence identity with AgraGR64f of Anthonomus grandis is 30.96%.qRT-PCR result showed that there were significant differences in the expression levels of EscrGR 8 in different developmental stages and adult tissues of E.scrobiculatus.EscrGR 8 had the highest expression level in female adults,and the lowest expression level in eggs.EscrGR 8 was highly expressed specifically in the female ovaries.

关 键 词：沟眶象 产卵量 味觉受体基因 RNAI 表达模式 

分 类 号：Q966[生物学—昆虫学]