【作者】 任爽；

【导师】 王国秀；

【作者基本信息】 华中师范大学 ， 动物学， 2011， 硕士

【摘要】 中华卵索线虫(Ovomermis sinensis)作为一种珍贵的昆虫天敌资源,在农林和蔬菜等害虫生物防治方面具有很好的应用前景。该类索科线虫的性别分化较为特殊,其寄生期的营养竞争压力决定线虫雌性性别的分化,即当寄生期幼线虫在昆虫体内的感染强度越低、每条线虫在血淋巴中获得的营养越多,越有可能发育成雌虫,反之则为雄虫。近年来,由于环境的破坏以及农药和化肥的滥用,外加索科线虫资源有限,这类生防资源正面临濒临消失的危险。国内外有关学者试图利用体外培养等方法解决现存问题,但未获成功,究其原因主要在于体外培养的线虫性别未能分化成熟,因而探讨索科线虫性别分化的机理成为本研究领域的重点。该领域研究的突破不仅具有非常重要的理论意义,而且具有潜在的应用价值,将为中华卵索线虫大量体外培养获得成功提供科学依据,并促使其尽快用于生产实际。DEAD-box家族作为一个ATP依赖RNA解旋酶家族,包含有四个亚家族成员,对生物体的生殖发育、器官分化以及细胞增殖等多个方面都具有重要作用。本实验室的前期工作已成功获得了这一家族的laf-1基因,并对其mRNA在中华卵索线虫不同发育时期的表达进行了检测,发现其在中华卵索线虫性腺发育的重要时期高水平表达,而其他时期的表达量都相对较低,由此推测这一基因在线虫的生殖腺发育中发挥作用。此外,利用荧光定量PCR的实验方法检测中华卵索线虫不同组织部位laf-1的表达,发现这一基因的转录本在多处组织部位都有表达,其功能可能较为广泛。同时获得的Tra-1基因,研究发现这一基因转录本在雌性成虫中表达量明显高于其他时期,推测其在雌虫生殖腺发育中具有重要作用。中华卵索线虫存在复杂的性别分化机理,对分子机理的深入探讨需要更多相关基因的研究支持。DEAD-box家族中的其他基因,如vasa、p68基因在从酵母到人类很多物种中都得到了相关研究。其中vasa基因常作为生殖细胞的分子标记用于原始生殖细胞的发生、迁移及发展等与生殖相关的研究；p68基因的转录本在很多物种的不同组织中都广泛检测到表达信号,这一基因的表达对生物体的生长发育同样起着非常重要的作用。而DEAD-box家族中的vasa、p68等基因在中华卵索线虫体内是否存在,这些基因在索科线虫中是否也像其他物种一样与生殖腺的发育相关联,这些基因在中华卵索线虫的哪些组织部位表达,表达是否具有特异性。为回答以上这些问题,本文在已有工作基础上进行了以下研究。一、采用RACE以及RT-PCR,获得了中华卵索线虫vasa基因,长度为3053bp,其中开放阅读框(ORF)2439bp,编码813个氨基酸。RT-PCR结果显示,vasa mRNA在中华卵索线虫的精巢、卵巢中特异性表达,而在其他部位,如头、尾、体壁均未检测到vasa的表达信号。vasa基因的转录本只在线虫的雌雄生殖腺中表达这一结果表明该基因与其生殖腺的功能相关,在线虫的精巢、卵巢发育过程中起发挥作用。二、运用实时荧光定量PCR,检测vasa mRNA在线虫发育不同期的表达。结果表明,vasa mRNA在胚胎时期表达量较高,在精巢和卵巢增殖的初期表达量逐渐增高,且在线虫从宿主体内脱离第5天达最高值(P<0.05)。随着性腺发育的逐渐成熟,其表达量减少。vasa mRNA早期胚胎发育过程中的表达显示,在中华卵索线虫胚胎发育早期都可以检测到vasa的转录信号,且相对表达量整体呈现先上升后下降的趋势。推测其为母源性基因,母源因子在个体胚胎发育中起了至关重要的作用。在线虫性腺发生的过程中,这一基因的转录信号最为强烈,在从宿主体内脱出之后,转录本显示出先上升之后下降的表达趋势,推测vasa与性腺的发生和分化相关联。三、利用RACE技术获得的中华卵索线虫p68(Ddx5)基因全长为2123bp,推测的氨基酸序列具有典型的DEXDc和HELICc结构域。四、运用RT-PCR方法,检测p68在中华卵索线虫不同组织部位,结果显示p68在线虫的不同部位都有表达,并且精巢、卵巢中的表达量较其他部位更为强烈。据报道,在鼠的雄性生殖细胞中P68表达水平很高,金鱼的p68基因在卵子及精子发生过程中发挥重要作用。因此推测该基因与生殖腺功能相关,同时还参与其他多种生理功能。更多还原

【Abstract】 Ovomermis sinensis is an important natural enemy of insects and has a great potential in biological controls of pests in the agricultures, vegetables and forest. As the Mermithidae of insect parasite, it has a specific sexual decision mechanism, which is determined by quantity of the nutrient through the parasitic stage. As environmental pollution, human destruction, and the limited resources of Mermithidae, these of bio-control resources nearly disappeared. Therefore, many scientists tried to culture the Mermithidae in vitro in order to solve the problem. However, this method is not successful, the reason is that the Mermithidae cultivated in vitro is not maturation in sex. We have known a little about the molecular mechanism of sex-determination about Mermithidae by now. On the other hand, Mermithidae is a valuable bio-control resource. Thus it is important for us to research the sexual decision mechanism of O. sinensis. For one thing, this research has the theory significance. For another thing, in order to carry out the culture in vitro of O. sinensis and provide insect enemy resources for the agricultural production, this study has the practice significance.The DEAD-box family genes vasa and p68 have been studied in many species from yeast to human. As the germ cell marker, vasa always use for the research of reproduction such as the production and removal of primordial germ cell and so on. The production signals of p68 mRNA are always detected in different tissues among distinct species. DEAD-box family proteins are the ATP dependent RNA helecases that contain four sub-families; they all involve in many aspects of the reproduction, developments, organ differentiation and cell proliferation. Predecessors in our lab have cloned laf-1 gene in O. sinensis and detected the expression of laf-1 mRNA through the all developmental periods. The results revealed that the signals were much higher during the sex determination period compared with other periods. In addition, the laf-1 mRNA has been detected in different tissues, the consequence manifested that the expression signals were appeared in many tissues. We have cloned Tra-1 which has essential function for the gender development in female adults.We have not understood whether the other genes of DEAD-box family exist and the roles of these genes in O. sinensis. In addition, O. sinensis has complex sex differentiation molecular mechanism. In order to research the mechanism of Mermithidae, we need more support of gene research. Therefore, to understand the sexual determination and reproduction of Mermithidae, we studied this special mechanism in molecular field as followings.1. Through the methods of RACE and RT-PCR, we have cloned the vasa of O. sinensis, which are 3053 base pairs for the full length. Its ORF consists 2439bp, and it encodes 813 amino acids. The results by the RT-PCR through the specific primers showed that vasa mRNA was expressed specifically in the gonads of both testis and ovary. However, in other tissues such as head, tail and body wall, there is no vasa mRNA expression signals.2. We have detected the expression of vasa mRNA through the defferent developmental stages of O. sinensis by Real Time PCR. The outcome showed vasa mRNA expression was high in embryo period, inceased firstly during the proliferation of testis and ovary, and was the highest during the fifth day of extrusion from the host(P< 0.05). It was decreased as testis and ovary matured. After leaving the host, the trends of the transcription signals showed that rise firstly, and then decline, so we speculated that vasa had the relationship with gonads development. The analysis results of early embyo development in O. sinensis showed that vasa mRNA was expressed during the all early embyo stages, and the relative expression increased firstly and decreased finally. It suggests the vasa of O. sinensis is a maternal gene and plays an essential role during early embryo development.3. The full length of p68 (Ddx5) of O. sinensis which is 2123bp was obtained by the method of SMART RACE. The putative amino acid contained the domains of DEXDc and HELICc.4. Through the method of RT-PCR, we have detected the expression of p68 mRNA in different tissues such as head, tail, testis, ovary and body wall in O. sinensis. Earlier reports showed that P68 was expressed highly in mice male germcells. The results showed that p68 gene was expressed in all these parts, we speculated that the activity of RNA helicase has functioned widely.更多还原