【作者】 李法财；

【导师】 胡敏；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2014， 博士

【摘要】 捻转血矛线虫(Haemonchus contortus)是寄生于反刍动物皱胃的寄生线虫,呈全球性分布,感染严重可导致幼畜的死亡,造成巨大的经济损失。由于缺乏有效的疫苗,针对寄生线虫病的控制主要依赖于抗蠕虫药,但是长期过度的使用抗蠕虫药已经造成抗药性的产生以及动物产品及环境药物残留问题。新药物和有效疫苗的研发因此越来越受到关注,而这必须对线虫的分子生物学有深入的了解。通过选择寄生线虫生活史中的关键过渡阶段作为研究对象,不仅能够帮助了解其生长发育的分子调控机制,而且也为防控寄生线虫病提供理论依据。一些重要的寄生线虫(包括猪蛔虫、马来丝虫、犬恶丝虫、捻转血矛线虫、罗拉丝虫和美洲板口线虫)在基因组和转录组取得的进展为分子生物学的研究提供了很好的平台。然而,体外培养和有效的基因操作技术缺乏严重阻碍了基因功能的研究。和秀丽新杆线虫(C. elegans)存在类似的滞育阶段(dauer),两者在生物学上存在诸多的相似性。首先秀丽新杆线虫同属于第五家族与H.contortus亲缘关系近,对外界环境有很强抵抗性,dauer可存活几个月并可以恢复发育完成生活史,与之相似,毛圆线虫(Strongylid)也存在类似的发育阶段(iL3),对外界环境有很强抵抗力,不摄取食物可存活数月。基于生物学上的相似性,一些学者提出了‘’dauer hypothesiS"(?)"Daf-c’’假说,认为寄生线虫三期幼虫阶段和C.elegans的dauer由相似的分子调控机制共同调节。C. elegans作为模式生物,对其发育生物学和分子生物学的研究处于前沿。研究发现dauer机制主要由胰岛素样信号通路调控,包括胰岛素样受体DAF-2,磷脂酰肌醇3-激酶AGE-1和AAP-1,3-磷脂酰肌醇依赖性蛋白激酶PDK-1和叉头转录因子DAF-16等。对daf-2,age-1和pdk-1突变会导致daf-c表型的出现,突变daf-16则是daf-d表型。信号通过DAF-2以磷酸化的方式激活AKT-1/2,再负反馈调节DAF-16,在调节生命周期、生长发育和抗逆性中发挥重要的功能作用。尽管对秀丽新杆线虫的dauer调控机制有较深入地了解,但对大多数寄生线虫而言,其发育调节几乎是一无所知。因此,本研究通过功能基因组学的方法分离了可能对H. contortus的发育发挥作用的Hc-daf-2.Hc-age-1、Hc-aap-1和Hc-pdk-1四个基因；RNAseq方法分析了基因在H. contortus的各个发育阶段的转录水平变化；异源转基因方法分析了基因启动子在秀丽新杆线虫的表达模式,基因救援法分析了基因的功能特点,从而在分子水平上对H. contortus的滞育发育(iL3)有更加深入的认识。(1)胰岛素样受体(Hc-DAF-2)结构与功能的研究采用兼并引物的方法分离Hc-daf-2基因的EST序列,在此基础上通过RACE, Genome Walker和Long-range PCR获得全长cDNA,gDNA和上游启动子序列。Hc-DAF-2与DAF-2家族基因高度同源,存在保守的结构功能域,并且存在与人胰岛素受体一致的蛋白水解位点(RKRR),推测与形成胰岛素受体复合物相关。Hc-daf-2在各个发育阶段都可转录,在感染性的L3期表达丰度明显上调。Hc-daf-2和Ce-daf-2启动子的表达模式,在C. elegans头感器神经位置表达具有相似性。另外,通过基因救援法发现Hc-daf-2基因对C. elegans daf-2缺失株(CB1370)能够呈现部分救援的效果。(2)磷脂酰肌醇3-激酶(PI3Ks)结构与功能的研究编码PI3K两个亚单位的基因Hc-age-1和Hc-aap-1与AGE-1和AAP-1家族具有很高的同源性,并且具有保守的结构功能区。转录水平分析发现Hc-age-1口Hc-aap-1基因在各个发育期都表达,在虫卵期、感染性L3期和雌性成虫期表达上调。启动子的表达模式研究,发现Hc-age-1和Hc-aap-1启动子驱动GFP在C.elegans肠道表达丰富,而Ce-age-1和Ce-aap-1在神经和肠道均有表达,更侧重于神经。在哺乳动物、果蝇和C.elegnns中,AGE-1和AAP-1会结合成二聚体构成PI3K激酶,为了研究Hc-age-1和Hc-aap-1是否会发生聚合,通过酵母双杂交的方法发现两者在酵母系统中能够高亲和力结合。基因救援法分析发现Hc-age-1基因对C.elegans age-1缺失株CY246没有呈现救援作用。(3)3-磷脂酰肌醇依赖性蛋白激酶(Hc-PDK-1)结构与功能的研究Hc-pdk-1与Ce-pdk-1基因有很高的同源性(46%),存在保守的功能区激酶催化区和PH区。PH区是一大类蛋白的功能调节区,在细胞信号转导以及细胞骨架功能中有重要作用。转录水平分析发现Hc-pdk-1在各个发育阶段均表达。转基因分析Hc-pdk-1和Ce-pdk-1基因启动子表达模式,Hc-pdk-1启动子表达在C.elegans的肠道组织,Ce-pdk-1主要表达在头部神经,咽部和肠道,两者在肠道部位存在一定的相似性。总之,这些发现有助于在分子水平上了解H.contortus发育的调控机制,特别是对从自由生活阶段向寄生生活的过渡阶段的调控作用。更多还原

【Abstract】 Haemonchus contortus is an abomasum-infected parasitic nematode that can lead to important disease of grazing sheep worldwide, causing weight reduction, death and enormous economic loss. As there is no effective vaccine available, treatment of parasitic nematode infections relies heavily on anthelmintics. However, the widespread use of these anthelmintics has resulted in serious resistance and drug residue problems worldwide. Therefore, it is imperative to develop new intervention strategies. One of the possibilities is the rational design of anti-parasite drugs and/or vaccines, built on the deep understanding of the biological and developmental processes in these parasites. For instance, for parasitic nematodes, clear insights into the developmental transition from free-living to parasitic stages might identify key switches as new drug targets.The characterization of the nuclear genomes and transcriptomes of some key parasitic nematodes, including Ascaris suum, Brugia malayi, Dirofilaria immitis, Haemonchus contortus, Loa loa and Necator americanus, provides a solid foundation for investigating developmental processes using complementary molecular (i.e. genetic, genomic, proteomic and metabolomic) tools. However, a lack of effective genetic and genomic tools for some parasitic nematodes and an inability to maintain their entire life cycle in the laboratory hampers detailed functional studies. In contrast, Caenorhabditis elegans can be readily maintained in the laboratory and used to explore fundamental processes and mechanisms, such as dauer formation. This free-living nematode belongs to clade V and is relatively closely related to H. contortus. Published information also indicates similarity in dauer induction and recovery between C. elegans and strongylids. The dauer state occurs in C. elegans when the nematode encounters unfavorable environmental conditions, including high temperature, starvation and/or crowding. The dauer form can survive for several months and resume development to reproductive adults when the environmental conditions improve. Consistent with C. elegans, strongylid nematodes have a similar infective L3(iL3), which is resistant to unfavorable conditions and does not feed because it is enveloped by a sheath. The "dauer hypothesis" or "daf-c paradigm" holds that the resumption of iL3development in parasitic nematodes is developmentally and functionally analogous to the recovery from dauer in C. elegans and is governed by similar molecular mechanisms. Dauer development in C. elegans is regulated by several signalling pathways, including an insulin/IGF-1-like signaling pathway, which contains a number of components, such as the insulin-like receptor kinase DAF-2, the PI3kinase AGE-1,3-phosphoinositide dependent protein kinase PDK-1and the FOXO-class transcription factor DAF-16. Mutations in the daf-2, age-1and pdk-1genes result in dauer constitutive (daf-c) phenotypes, whereas mutations in daf-16give dauer-defective (daf-d) phenotypes. Signalling through DAF-2activates AKT-1/2by phosphorylation, which, in turn, negatively regulates DAF-16, which functions as a central mediator of multiple biological processes, including longevity, development and stress resistance.(1) The structural and functional characterizations of the insulin-like receptor(Hc-daf-2)Using a PCR-based approach, we identified and characterized a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2displays significant sequence homology to insulin receptors in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2is consistent with that of the human insulin receptor (HIR), suggesting that it is involved in the formation of the insulin-receptor complex. The Hc-daf-2gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the infective third-stage (iL3) compared with other stages. To compare patterns of expression between Hc-daf-2and Ce-daf-2, reporter constructs fusing the Ce-daf-2or Hc-daf-2promoter to sequence encoding the green fluorescent protein (GFP) were micro injected into the N2strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2.(2) The structural and functional characterizations of the phosphatidykinositol3-kinasePI3K (Hc-AGE-1and Hc-AAP-1)We identified and characterized the PI3K catalytic subunit (Hc-AGE-1) and regulatory subunit (Hc-AAP-1) from Haemonchus contortus. The sequence of Hc-AGE-1and Hc-AAP-1show high homology to PI3K in both vertebrates and invertebrates, and contain conserved structural domains. The Hc-age-1and Hc-aap-1genes were transcribed in all life stages of H. contortus, with up-regulation in the eggs, infective third-stage (iL3) and female adults compared with other stages. To compare patterns of expression between Hc-age-1, Ce-age-1and Hc-aap-1, Ce-aap-1, reporter constructs fusing the promoter to sequence encoding the green fluorescent protein (GFP) were microinjected into the N2strain of C. elegans, respectively, and transgenic lines were established and examined. Hc-age-1and Hc-aap-1show the expression pattern in intestine, and Ce-age-1and Ce-aap-1display in intestine and neurons. Yeast two-hybrid system showed the p85binding domain of Hc-age-1and Hc-aap-1can interact with each other strongly. Further study by heterologous genetic complementation in an oge-1-deficient strain of C. elegans (CY246) showed no rescue of function by Hc-age-1.(3) Characterizations of the3-phosphoinositide dependent protein kinase (Hc-PDK-1)We identified and characterized the3-phosphoinositide dependent protein kinase from Haemonchus contortus. The Hc-PDK-1shows high homology with Ce-PDK-1(about46%), and contains catalytic kinase domain and PH domain. PH domain is a functional regulator of a group of protein kinases and plays a center role in cell signalling transduction and cytoskeletal function. The Hc-pdk-1genes were transcribed in all life stages of H. contortus. To compare patterns of expression between Hc-pdk-1and Ce-pdk-1, reporter constructs fusing the promoter to sequence encoding the green fluorescent protein (GFP) were microinjected into the N2strain of C. elegans, respectively. The expression location of Hc-pdk-1is in intestine, and Ce-pdk-1is in head neurons, pharynx and intestine.Taken together, these findings provide a first insight into the roles of Hc-daf-2/Hc-DAF-2, Hc-age-1/Hc-AGE-1, Hc-aap-1/Hc-AAP-1and Hc-pdk-1/Hc-PDK-1in the biology and development of H. contortus, particularly in the transition to parasitism.更多还原