【作者】 张琼；

【导师】 李国清；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2019， 硕士

【摘要】 昆虫蜕皮激素(molting hormone,MH)是甾醇类激素,在昆虫体内的活性形式为20-羟基蜕皮酮(20-hydroxyecdysone,20E)。蜕皮激素与蜕皮激素受体(ecdysone receptor,EcR)和超气门蛋白(ultraspiracle,USP)组成的复合体结合并相互作用,启动20E级联信号转导途径,调控昆虫的生长发育、繁殖以及先天免疫等重要的生理学过程。本文根据马铃薯甲虫(Leptinota decemlineata)转录组和基因组数据,采用基因克隆技术和生物信息学方法,获得了马铃薯甲虫蜕皮激素受体EcR基因的2条剪切体的cDNA全长序列。构建干扰载体dsLdEcRA和dsLdEcRB1;RNA干扰沉默该基因,观察对幼虫发育、化蛹和羽化的影响以及LdEcRA、LdEcRB1在20E信号通路中的作用,取得了如下结果。1.蜕皮激素受体EcR基因的分子克隆以及进化分析基于马铃薯甲虫转录组和基因组数据,鉴定出蜕皮激素受体EcR基因的2条剪切体cDNA,利用聚合酶链式反应(PCR)技术,获得这2个EcR剪切体基因的全长cDNA序列,这些剪切体编码的蛋白都具有典型的核受体家族结构特征。根据与赤拟谷盗、黑腹果蝇等其他物种进化分析结果,将2个EcR剪切体分别命名为LdEcRA和LdEcRB1。这2个EcR剪切体与赤拟谷盗的相关基因对应,表明我们鉴定出了马铃薯甲虫全部EcR基因剪切体。2.LdEcRA 和 LdEcRB1 的时空表达分析LdEcRA和LdEcRB1在不同发育期的表达,发现它们在马铃薯甲虫的整个生育期内均表达。其中LdEcRA在四龄幼虫第4天达到表达的最高水平,而LdEcRB1在四龄初大量表达。组织表达谱分析结果发现,LdEcRA和LdEcRB1在四龄幼虫的脑-心侧体-咽侧体、前胸腺、中肠、后肠、脂肪体以及表皮中均有较高的表达;其中LdEcRA的表达量大于LdEcRB1。3.干扰LdEcR基因对幼虫生长发育的影响使用大肠杆菌表达3种dsRNA:其中dsEcR源于LdEcRA和LdEcRB1的共有段,而dsEcRA和dsEcRB1则分别源于LdEcRA和LEcRB1的特有序列。饲喂3龄和4龄幼虫dsEcR和dsEcRA均影响幼虫到蛹、以及蛹到成虫的蜕皮;但饲喂dsEcRB1则无明显影响。取食了 dsEcR和dsEcRA的幼虫停止发育、或变为畸形的蛹和成虫。比较分析不同处理幼虫中EcR的mRNA水平发现,LdEcRA控制LdEcRB1的表达。当LdEcRA和LdEcRB1两个剪切体均被干扰,蜕皮激素合成酶基因 Ldspo和Ldsad的转录水平和20E滴度显著降低。20E信号途径基因HR3、HR4、E74和E75的mRNA水平在LdEcR或LdEcRA沉默的幼虫中显著下降,但在LdEcRB1干扰的幼虫中保持不变。沉默LdEcR或LdEcRA的幼虫即使取食20E也不能恢复USP、HR3、HR4、E74和E75的转录水平,仅轻微减缓RNA干扰对化蛹和羽化的影响。以上结果表明LdEcRA和LdEcRB1对马铃薯甲虫幼虫化蛹和羽化的调控具有剪切体特异性。更多还原

【Abstract】 The molting hormone in insects is a steroid,and its active form is 20-hydroxyecdysone(20E).20E exerts its effects through a heterodimer of ecdysone receptor(EcR)and ultraspiracle(USP).The 20E/EcR/USP complex binds to ecdysone response elements to activate early 20E-response genes.In insects,20E modulates many aspects in the life,such as reproduction,embryogenesis,molting and metamorphosis,diapause and innate immunity.Based on the transcriptome and genome data of the Colorado potato beetle Leptinotarsa decemlineata,the full-length cDNA sequences of two ecdysone receptor isoforms(LdEcRA and LdEcRB1)were obtained.DsLdEcR(targeting both isoforms),dsLdEcRA and dsLdEcRB1 were constructed.The roles of LdEcRA and LdEcRB1 in 20E signaling pathway and the effects of silencing these isoforms on larval development,pupation and emergence were investigated.1.Molecular cloning of EcR isoforms in Leptinotarsa decemlineataBased on the transcriptome and genome data of L.decemlineata,two EcR isoforms were identified.The full-length cDNA sequences were obtained by PCR technology.Both proteins encoded by these two isoforms had typical structural characteristics of nuclear receptor families.The two EcR isoforms were named LdEcRA and LdEcRB1 by the evolutionary analysis with other insect species,such as Tribolium castaneuma and Drosophila melanogaster.These two EcR isoforms are corrsponded to the ralated splicing variants in Tribolium castaneuma,which indicate that we have identified all EcR isoforms in L.decemlineata.2.Spatiotemporal expression of LdEcRA and LdEcRBl in L.decemlineataThe expression patterns of LdEcRA and LdEcRB1 were analyzed in different development stages and different tissues.LdEcRA and LdEcRB1 transcripts were detectable from the egg to adult.LdEcRA was abundantly expressed in the final larval instars,wandering prepupae,pupae and adults.It peaked at day 4 final instar stage.In contrast,LdEcRB1 was mainly transcribed from the embryo(eggs)to day 1 final instar larvae and in the pupae.Its peak appeared just after the molt of the final instar larvae.The tissue-specific expression patterns of LdEcR isoforms were also tested by qRT-PCR.The results clearly showed that both isoforms were expressed in the brain-corpora cardiaca-corpora allata complex,prothoracic glands,foregut,midgut,hindgut,Malpighian tubules,fat body and epidermis of the day 4 final instar larvae.In addition,the expression level of LdEcRA was higher than that of LdEcRB1.3.The influence of LdEcR isoforms on the growth and development of larvaThree dsRNAs were expressed by Escherichia coli.Out of these dsENAs,dsLdEcR was derived from the common segment of LdEcRA and LdEcRB1,while dsLdEcRA and dsLdEcRBl were come from the specific segment of LdEcRA and LdEcRB1,respectively.Feeding dsEcR or dsEcRA,rather than dsEcRB1,caused the failure of pupation and adult emergence.Larvae feeding on dsLdEcR or dsEcRA arrested in development or became deformed pupae and adults.Determination of mRNA levels of EcR isoforms found that LdEcRA regulates the expression of LdEcRB1.Moreover,silencing the two EcR transcripts,LdEcRA or LdEcRB1 reduced the mRNA levels of Ldspo and Ldsad,and lowered 20E titer.In contrast,the expression levels of HR3,HR4,E74 and E75 were significantly decreased in the LdEcR or LdEcRA RNAi larvae,but not in LdEcRBl depleted specimens.Dietary supplement with 20E did not restore the expression of five 20E signaling genes(USP,HR3,HR4,E74 and E75),and only partially alleviated the pupation defects in dsEcR-or dsEcRA-fed beetles.The results indicated that EcR is undoubtedly necessary for regulation of ecdysteroidogenesis and for mediation of 20E signaling,in an isoform-dependent pattern,in L.decemlineata.更多还原