拟黑多刺蚁蜕皮激素受体（EcR）和超气门蛋白（USP）的cDNA测序、mRNA表达及其与雌激素相关受体（ERR）的功能相关性分析

【作者】 刘晓明；

【导师】 奚耕思；

【作者基本信息】 陕西师范大学 ， 发育生物学， 2013， 博士

【摘要】 蜕皮激素受体(ecdysteroid receptor,EcR)是昆虫中唯一一个配体已知的核受体,它的配体为蜕皮激素(ecdysteroid hormones, EH)。昆虫生长发育过程中,蜕皮和变态等生理过程都受到蜕皮激素的严格调控,EcR是蜕皮激素的作用靶标,处于昆虫变态、蜕皮及繁殖等生命过程级联反应的启动位置,对于完成昆虫生长发育及繁殖过程具有十分重要的作用。超气门蛋白(ultraspiracle protein, USP)隶属于核受体第二亚家族,与保幼激素具有亲和性,是保幼激素受体的候选基因,在昆虫的蜕皮变态过程中,蜕皮激素受体必须与超气门蛋白结合形成异源二聚体后才能与蜕皮激素结合,激活下游蜕皮反应基因,引起蜕皮变态的级联反应。雌激素相关受体(estrogen receptor-related receptor, ERR)是人类发现的最早的孤儿核受体之一,在结构上它与雌激素受体(estrogen receptor, ER)非常相似,对哺乳类动物的研究表明,ERRs参与ER信号通路,与众多生长发育过程密切相关,并且参与细胞的增殖与分化,但该基因不能与雌激素相结合。在昆虫中也发现ERR的存在,但是其具体作用仍然不是很清楚。拟黑多刺蚁(Polyrhachis vicina Roger)隶属于昆虫纲(Insecta)、膜翅目(Hymenoptera),蚁科(Formicidae),多刺蚁属(Polyrhachis),是一类典型的营群居性生活的社会性昆虫,同时,也是国家卫生部指定的药食兼用型资源昆虫。拟黑多刺蚁具有社会性昆虫的典型特征,具有广泛的多态现象和高度复杂的神经系统,是研究昆虫发育的优良动物实验材料。本文以拟黑多刺蚁为实验材料,通过RT-PCR技术、RACE末端扩增技术、荧光实时定量RT-PCR技术、原位杂交技术以及RNA干扰技术对拟黑多刺蚁蜕皮激素受体(EcR)、超气门蛋白(USP)及雌激素相关受体(ERR)基因进行克隆、表达定位定量及功能相关性分析,研究结果如下：1.拟黑多刺蚁EcR基因cDNA序列全长2235bp,包含一个长度为1737bp的开放阅读框,5’-UTR为20bp,3’-UTR为478bp。将序列命名为PvEcR,上传至GenBank,获得序列号为JX028426.1。PvEcR蛋白质含有579个氨基酸残基,分子量为63.4kDa,理论等电点pI=7.79,为亲水性蛋白质,N端不存在信号肽,为非分泌性蛋白。PvEcR蛋白氨基酸序列与膜翅目昆虫日本弓背蚁、大头蚁、无刺蜂、熊蜂和意大利蜜蜂的序列相似性高达99%,与其他昆虫类的蛋白序列相似性也很高,基本在70%以上。蛋白质二级结构分析表明PvEcR蛋白是alpha-beta型。利用实时定量PCR方法研究PvEcR基因在拟黑多刺蚁的不同发育阶段、三个品级及脑部的表达量,结果表明,在拟黑多刺蚁的幼虫期,PvEcR的表达量较高,特别是在3龄幼虫期,PvEcR的表达量达到最高,随后,在4龄幼虫期缓慢下降,在蛹期的PvEcR的表达量急速下降,至成虫期,表达量又缓慢恢复。在拟黑多刺蚁的三个品级成虫中,PvEcR的表达量没有太大的差异,但是在不同品级的成虫头部中,雄蚁头部的PvEcR表达量最高,推测该基因可能跟雄性蚂蚁特定的神经系统以及雄特有的行为相关。利用荧光原位杂交技术对PvEcR在拟黑多刺蚁三个品级头部的表达进行定位分析,结果显示PvEcR广泛表达于拟黑多刺蚁不同品级脑部,且表达量存在差异。PvEcR在工蚁和雌蚁脑部蕈形体中高表达,特别是在球形细胞中阳性表达较高,在雄蚁脑部的视叶中,PvEcR阳性反应较其它两品级显著,说明PvEcR可能与蚂蚁神经系统调控的趋光性、求偶行为相关。利用RNA干扰技术抑制PvEcR在拟黑多刺蚁不同发育阶段幼虫、不同品级成虫的表达,结果表明,在蚂蚁的胚胎期及1龄幼虫期,该基因的表达量无明显变化,自2龄幼虫开始,PvEcR表达量开始下降,特别是在PvEcR高表达的3龄、4龄幼虫期,该基因表达量开始下降,并引起昆虫表型的变化,出现幼虫体重降低、成虫雄蚁残翅等现象,说明我们所使用的dsRNA测试液可以引起该基因的沉默现象,也证明了PvEcR在蚂蚁发育阶段及成虫翅膀发育过程中的重要作用。2.拟黑多刺蚁USP基因cDNA序列全长1553bp,包含一个长度为1275bp的开放阅读框,5’-UTR为78bp,3’UUTR为200bp,将序列命名为PvUsP.上传至GenBank,获得序列号为KC188780.PvUSP含有424个氨基酸残基,蛋白质分子量为47.7kDa,理论等电点pi=8.63,为亲水性的蛋白质,N端不存在信号肽,为非分泌性蛋白。PvUSP与膜翅目各类蜜蜂的USP均有80%以上的序列相似性,与其它昆虫的序列相似性在70%以上。蛋白质二级结构分析表明PvUSP蛋白是alpha-beta型。通过荧光实时定量RT·PCR分析jvUSP在拟黑多刺蚁不同发育阶段虫体、不同品级成虫及头部的表达情况,结果表明PvUSP基因在所检测的各个时期及各个品级内均有表达。PvUSP高表达于拟黑多刺蚁胚胎期,说明其在胚胎发生过程和细胞增殖分化中有重要作用。在幼虫的发育过程中,PvUSP在蛹期存在一个表达高峰,该高表达一直持续到昆虫变态完成,说明该基因在昆虫的变态发育中具有重要作用。在拟黑多刺蚁的三个不同品级成虫中,PvUSP在雄蚁中的表达量最高,工蚁次之,雌蚁最少,在三个品级成虫头部,PvUSP在工蚁头部的表达量最高,雄蚁次之,雌蚁最少,PvUSP在三个品级成虫及头部的不同表达趋势证明该基因参与了拟黑多刺蚁中神经系统的构建,并在雄性生殖系统中发挥作用。3.利用荧光实时定量RT-PCR分析PvERR在拟黑多刺蚁不同发育阶段虫体、不同品级成虫及头部的表达情况,结果表明PvERR在拟黑多刺蚁发育过程中的表达量具有阶段特异性。在幼虫的胚胎期和1龄、2龄等蚂蚁早期发育过程中,PvERR的表达量基本恒定,自3龄幼虫开始,PvERR的表达量逐渐增加,在成虫的表达量最高,说明PvERR与拟黑多刺蚁的发育有关。PvERR在拟黑多刺蚁蛹期到成虫期相对表达量有了明显增加,这可能说明PvERR在成虫的组织分化中具有重要作用。在拟黑多刺蚁的三个品级中,PvERR在雌蚁中的表达量远远高于工蚁和雄蚁,说明该基因可能与雌蚁生殖功能相关。在拟黑多刺蚁三个品级成虫和脑部中,PvERR在雌蚁中表达量最高,工蚁次之,雄蚁最少,该基因在不同品级间的表达差异可能与它们的社会分工相关,关于这方面的内容,还需要进一步的研究。利用荧光原位杂交技术对PvERR在拟黑多刺蚁三个品级头部的表达进行定位分析,结果表明PvERR在拟黑多刺蚁不同品级脑部蕈形体和球形细胞中的阳性表达最为强烈,说明该基因参与了拟黑多刺蚁神经系统的发育。在拟黑多刺蚁的不同品级脑部,PvERR的表达量各不相同,说明该基因的具体功能与拟黑多刺蚁的社会分工相关。利用RNA干扰技术抑制PvERR在拟黑多刺蚁不同发育阶段幼虫、不同品级成虫体内的表达,结果表明,在蚂蚁的胚胎期及1龄、2龄幼虫期,该基因的表达量无明显变化,自3龄幼虫期至成虫期,PvEcR表达量下降得较为明显,下降幅度在50%-60%之间。这说明我们所用的dsRNA测试液能够沉默PvERR的表达,并引起幼虫及成虫体重降低、3龄和4龄幼虫致死等现象,从而也证明了PvERR在蚂蚁的生殖和发育阶段具有重要作用。4.本研究利用RNAi干扰技术,研究PvEcR、PvUSP和PvERR在拟黑多刺蚁发育与生殖阶段的功能相关性分析。实验结果表明,在PvEcR的表达被沉默或表达量极少的时候,PvUSP能够在蜕皮过程关键的4龄幼虫期和蛹期提高表达,弥补PvEcR的表达缺失,与对照组相比,ERR的表达量虽有提高,但是表达量没有显著性差异,说明PvEcR的沉默不会引起PvERR的表达量变化；在PvERR表达被沉默或表达量极少时,PvEcR和PvUSP在蚂蚁发育前期表达量有下降的趋势,但是在蜕皮变态的蛹期,两基因都有不同程度的上升,特别是PvEcR的表达量上升尤为显著,因此,我们推测PvERR可能处于PvEcR和PvUSP的上游调控位置,但是其具体功能,还需要进一步深入探究。更多还原

【Abstract】 The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) and is the only ligand-depended nuclear receptors known in insects. Its ligand is ecdysteroid hormones (EH),which plays critical roles in control insect growthing, developmenting, molting as well as metamorphosising. EcR takes part in the signal path of EH, also, EcR is in the key possion of cascade process including insect metamorphosising, molting and reproduction.As a member of the second nuclear receptors subfamily,Ultraspiracle protein (USP) possesses a affinity with Juvenile hormone(JH),and is thought to be the candidate receptors of JH. During insect molting and metamorphosising,EcR must bind USP to form heterologous dimmers, then combine with EH,activate downstream of relation gene and cause cascade response in molt, metamorphosis as well as reproduction.The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the function and regulation of ERRs in invertebrates are not well understood.Polyrhachis vicina Roger belongs to the genus Polyrhachis (Hymenoptera:Formicidae). As a typical kind of eusocial insects, P.vicina possesses the characteristic of castes differentiation, sophisticated behaviors, behavioral plasticity and highly complex nervous system.In this study, the full-length cDNAs of P.vicina EcR and USP gene is coloned by RT-PCR and RACE methods. The bioinformatics method is used to analyze characteristics of full-length cDNAs and predict functional motifs in the ORF. The mRNA expression levels of EcR、USP and ERR in the ants were investigated by real-time RT-PCR and in situ hybridization methods. The functional relationship among the three genes were investigated by RNAi.The major experiment results are as followed:1. The full-length cDNA of EcR in P.vicina is2235bp, containing an open reading frame of1737bp, which encodes a deduced579-amino acid peptide with a predicted molecular mass of63.4kDa and with the theoretical p1=7.79,which contains a5’-untraslated region (5’-UTR) of20bp and a3’-UTR of478bp. The nucleotide sequence of EcR in P.vicina, named PvEcR, is submitted to GenBank and assigned the number JX028426.1. The results of homologous analysis showed that the full length cDNA of EcR in P.vicina is similar to Camponotus japonicus, Pheidole megacephala and Apis mellifera at99%,the similarity to other insects is over70%. Bioinformatics analysis showed that PvEcR protein belongs to a non-secreted member. In this study, a fluorescent real-time quantitative RT-PCR is used to analyze the relative quantification expression of the mRNA level of PvEcR in different developmental periods, different castes adults and different adults’brain. The results showed that PvEcR is expressed in each tested sample. The expression level of PvEcR in lavar is higher than other stages, the highest expression level is found in the third instar, and declined until the pupal. Among three caste adults, the expression of PvEcR is almost the same. The PvEcR mRNA expression analysis among the heads of the three casts showed that the expression level from height to low was male, worker and female ants in turn. The distribution of PvEcR mRNA was analyzed by in situ hybridization to cryosections of the brain of adult ants. The results showed that the hybridization signals are present extensively in the brains of all adult’s heads.The hybridization signals are highest expression in the mushroom bodies of worker and females.In male ants,the highest hybridization signals are found in the optic lobes and deutocerebrum. From these results,we speculate that the functions of PvEcR are involved in obtaining and integrating the visual and olfaction information in the nervous system.. Using RNAi technology to silence the expression of PvEcR mRNA in different developmental periods, different castes adults, the results showed that, in embryo and first instar stages, the expression of PvEcR has no obvious changes,from the second instar,the expression began to decline, especially in the stages of third and fourth instar, when the expression of PvEcR is much higher than others. The expression of PvEcR was significantly decreased, and leading to insect phenotypic changes containing larval weight loss, the residual wings of male ants, illustrate that in this study,the dsRNA solution we used can reduce the expression of PvEcR mRNA, also prove the important function of PvEcR during the process of development in ants.2. The full-length cDNA of USP in P.vicina is1553bp, containing an open reading frame of1275bp, which encodes a deduced424-amino acid peptide with a predicted molecular mass of47.7kDa and with the theoretica1=8.63,which contains a5’-untraslated region of78bp and a3’-UTR of200bp. The nucleotide sequence of USP in P.vicina, named PvUSP, is submitted to GenBank and assigned the number KC188780.The results of homologous analysis showed that the full length cDNA of USP in P.vicina is similar to bees at over80%, and to other insects is over70%. Bioinformatics analysis showed that PvUSP protein belongs to a non-secreted member. In this study, a fluorescent real-time quantitative RT-PCR is used to analyze the relative quantification expression of the mRNA level of PvUSP in different developmental periods, different castes adults and different adults’brain. The results showed that PvUSP is expressed in each tested sample. During the development stage, the highest expression level is found in embryo and pupa. Among three caste adults, the highest expression of PvUSP is male ants. The PvUSP mRNA expression analysis among the heads of the three casts showed that the expression level from height to low was worker, male and female ants in turn. From these results we speculate that the high mRNA levels in both the embryo and adults may suggest the essential role of PvUSP in ant development and the nervous system.3. In this study, a fluorescent real-time quantitative RT-PCR is used to analyze the relative quantification expression of the mRNA level of PvERR in different developmental periods, different castes adults and different adults’brain. The results showed that PvERR is expressed in each tested sample. During the development stage, the expression level of PvERR is gradually increased, and the highest levels are found in different castes, expecially in female ants. Among the heads of the three casts, the results showed that the expression level from height to low was female, worker and male ants in turn. The distribution of PvERR mRNA was analyzed by in situ hybridization to cryosections of the brain of adult ants. The results showed that the hybridization signals are present extensively in the brains of all adults’ heads.The hybridization signals are highest expression in the mushroom bodies of worker and females, in male ants, the highest hybridization signals are found in the optic lobes and deutocerebrum. From these results, we speculate that the functions of PvERR are involved in the visual information integration, further to control the courtship behavior. Using RNAi technology to silence the expression of PvERR mRNA in different developmental periods and different castes adults, the results showed that, in embryo, first instar and second instar stages, the expression of PvERR has no obvious changes, from the third instar to adults, the expression declined significantly, in each tested samples, the expression of PvERR mRNA may decreased from50%to60%. the weight of larval and adults was lost, some larval were dead in three and fourth instar, all the results illustrate that in this study the dsRNA solution we used can reduce the expression of PvERR mRNA, and also prove that the PvERR plays an important role in the reproductive and developmental stages of the ants.4. In this study, RNAi is used to analyze the functional relationship among the three genes during ants’ development. The results showed that when the expression level of PvEcR was silent or low, the expression level of PvUSP would be immediately increased to cover the lack of PvEcR’s expression; when the expression level of PvERR was silent or low, the expression level of PvUSP and PvEcR would be decreased during the larva stage,but in pupal and adult,both of the two genes’ expression would be increased, expecially the expression of PvEcR.From these results, we speculate that, ERR can regulate the expression of EcR and USP during the development of ants,but more information is needed to be further explored. In conclution we have cloned the full-length cDNAs of EcR and USP genes firstly from the ants, P. vicina, and submitted the cDNA sequences to GenBank. The results of real-time quantitative RT-PCR and in situ hybridization methods revealed that the PvEcR, PvUSP and PvERR mRNA were found in all developmental stages, different castes and heads. The functional relationship among the three genes ware analyzed by RNAi.This may indicate that three genes have the main physiological functions and involved in regulating of growth, development and reproduction in insects. These results may provide the theory foundation for further investigation on the concrete function of the three genes in insects.更多还原