日本血吸虫雌虫合抱前后差异表达基因的筛选与鉴定

发布时间：2018-04-08 12:40

本文选题：血吸虫雌虫　切入点：抑制性消减杂交技术　出处：《安徽医科大学》2014年硕士论文

【摘要】：目的：血吸虫病(schistosomiasis)是由血吸虫(Schistosoma)感染引起的一种分布广泛、危害严重的人畜共患寄生虫病。血吸虫为吸虫中罕见的雌雄异体形态特征,雌雄虫体之间的相互作用对血吸虫性器官的发育和产卵起着至关重要的作用,血吸虫雌虫性器官的发育成熟和产卵必须通过雌雄虫合抱这一生物学表象方可完成。成熟雌虫所产生的大量虫卵而引起的脏器病变是造成宿主主要病理损害和疾病传播的前提。本研究目的为筛选鉴定日本血吸虫雌虫在合抱前期(感染后16天)、合抱初期(感染后18天)和合抱后期(感染后24天)差异表达基因,确定其为雌虫生殖发育相关的关键分子,加深对合抱的血吸虫性发育成熟和产生活性虫卵的理解,为控制血吸虫病的流行和危害寻找新的靶标。方法：应用日本血吸虫感染模型,给以每只昆明鼠感染70条左右尾蚴,分别于14、15、16、17和18天剖杀小鼠,灌注法收集虫体,观察计数该时间段雌雄合抱的对数,确定血吸虫雌雄合抱前和雌雄合抱初期的具体时间。再应用抑制性消减杂交技术(suppression subtractive hybridization,SSH))筛选日本血吸虫雌虫在雌雄合抱前(感染后16天)、合抱初期(感染后18天)和合抱后期(感染后24天)的差异表达基因。先用合抱初期的雌虫cDNA作为检测子(tester),合抱前期的雌虫cDNA作为驱赶子(driver),检测子消减驱赶子富集合抱初期的高表达基因,驱赶子消减检测子富集合抱前期的高表达基因；再用合抱后期的雌虫cDNA作为检测子,合抱初期的雌虫cDNA作为驱赶子,检测子消减驱赶子富集合抱后期的高表达基因的,驱赶子消减检测子富集在合抱初期高表达基因,从而找出血吸虫雌虫合抱前、合抱初期以及合抱后期的差异表达基因。利用生物信息学分析工具对上述所有筛选出的差异基因所编码的蛋白进行GO功能分类和KEGG代谢通路分析,依据分析结果推测其中28个可能是参与雌虫生殖发育密切相关的基因,再用RT-qPCR (Real time quantity polymerase chain reaction)以不同发育期的雌虫cDNA为模板对该28个基因进行进一步的验证。从血吸虫雌虫中分离卵巢,提取卵巢总RNA,再分别卵巢cDNA和成虫从cDNA为模板,使用RT-qPCR观察这些与生殖发育相关基因在卵巢的表达量。结果：根据小鼠感染后不同时间血吸虫合抱发生状态的试验,确定血吸虫感染小鼠16天时为合抱前期,18天时为合抱初期,24天为合抱后期(性器官基本发育成熟)。用SSH技术成功构建了日本血吸虫雌虫合抱初期和合抱前期的正、反向消减文库,即相对于合抱前期的合抱初期高表达基因cDNA文库和相对于合抱初期的合抱前期高表达基因cDNA文库。同法还构建了血吸虫感染雌虫合抱后期与合抱初期的正、反向消减文库,即相对于合抱初期的合抱后期高表达基因cDNA文库和相对于合抱后期的合抱初期高表达cDNA文库。对相对于合抱前期的合抱初期高表达基因、相对于合抱初期的合抱前期高表达基因、相对于合抱初期的合抱后期高表达基因以及相对于合抱后期的合抱初期高表达基因进行测序,去除重复序列、序列拼接之后,分别获得112个、96个、195和172个高表达基因序列标签(Expressed Sequence Tag, EST),通过与数据库进行比对,得到有明确注释信息的分别有92、80、103和90个EST序列。对这些EST所编码蛋白的分子功能、细胞定位、参与的生物学过程进行基因本体论(Gene Ontology,GO)注释。相对于合抱前期的合抱初期高表达基因主要参与的生物学过程为环状化合物代谢过程、含氮化合物的代谢过程、细胞大分子生物合成过程、氧化-还原过程等。相对于合抱初期的合抱前期高表达基因主要参与的生物学过程包括含氮化合物的代谢过程、磷代谢过程、环状化合物代谢过程、小分子代谢、转运过程等。相对于合抱初期合抱后期高表达基因主要参与的生物学过程如转运、碳水化合物的代谢过程、核苷酸代谢、小分子代谢过程等。相对于合抱后期的合抱初期高表达基因主要参与的生物学过程包括转运,核苷酸代谢、磷代谢、跨膜转运等。使用京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)对相对于合抱前期的合抱初期高表达基因、相对于合抱初期的合抱前期高表达基因、相对于合抱初期的合抱后期高表达基、以及相对于合抱后期的合抱初期高表达基因编码蛋白所参与的信号通路进行分析。KEGG分析发现相对于合抱前期的合抱初期高表达基因和相对于合抱初期的合抱前期高表达基因所编码蛋白参与76条信号通路；从合抱后期高表达基因而相对于合抱初期的合抱后期高表达基因和相对于合抱后期的合抱初期高表达基因这所编码蛋白参与79条信号通路。多个高表达基因集中参与上述信号通路主要包括代谢通路,氧化磷酸化过程,内质网蛋白质加工过程,特别是发现了与生殖发育相关的信号通路高表达基因。在这些信号通路中筛选出28个可能与生殖发育信号通路相关的基因进行荧光定量PCR的进一步验证,结果18个基因经验正与消减杂交结果一致。其中有与生殖发育直接相关的参与泌乳素和雌激素信号通路的酪氨酸激酶(tyrosine kinases, Tks)、热休克蛋白70(Heat shock protein70,HSP70)和热休克蛋白90(Heat shock proteins90, HSP90)、TGF-β信号通路相关的Type V collagen蛋白、参与Wnt信号通路的早老素蛋白、参与酵母细胞的发育周期的NIPBL蛋白(Nipped-β-like protein)和参与Hippo信号通路中的mob蛋白。其中HSP70、HSP90和早老素蛋白(presenilin)在合抱后期的雌虫的基因水平明显上调,而酪氨酸激酶、Type V collagen蛋白、NIPBL蛋白和mob蛋白在合抱初期雌虫的基因水平明显上调。 RT-qPCR技术对雌虫生殖器官卵巢cDNA和雌虫成虫cDNA的Tyk、HSP90、 Type V collagen蛋白、早老素蛋白、NIPBL蛋白(Nipped-β-like protein)和mob蛋白的分析,结果显示NIPBL蛋白和早老素蛋白在卵巢高表达。结论：本实验通过抑制性消减杂交技术成功构建了日本血吸虫雌虫合抱前期、合抱初期、合抱后期的消减cDNA文库。筛选出7个可能直接与血吸虫雌雄合抱和产卵相关的分子包括Tks、HSP70和HSP90、Type V collagen蛋白、早老素蛋白、NIPBL蛋白、mob蛋白等。其中有的Tks、HSP70参与血吸虫生殖发育的功能已经有文献报道,Type V collagen蛋白、早老素蛋白、NIPBL蛋白、mob蛋白参与血吸虫生殖发育的功能尚有待进一步明确。
[Abstract]:Objective: schistosomiasis (schistosomiasis) by schistosome infection (Schistosoma) is a kind of distribution caused by widespread, serious zoonotic parasitic diseases. Schistosomiasis characterized dioecious fluke in rare form, the interaction between the male and female worms of Schistosoma mansoni organ development and oviposition plays a vital role in female Schistosoma japonicum organ maturation and spawning by male and female worms of the biological image to complete. Organ lesions in a large number of eggs produced by mature females is caused by the main pathological damage caused by the premise of host and the spread of the disease. The purpose of this study is to screening and identification of Schistosoma japonicum in a previous (16 days after infection), a early (18 days after infection) and a late (24 days after infection) of differentially expressed genes identified as female reproductive development related key points, deepen the The development of Schistosoma japonicum and the understanding of the production of active insect eggs will find new targets for the control of the epidemic and harm of schistosomiasis.
Methods: application of Schistosoma japonicum infection model, give each about 70 Kunming mice infected with cercariae, respectively at 14,15,16,17 and 18 days mice were killed, perfusion worms collected, observation log counting the time of pairing, determine the specific time of Schistosoma japonicum male femalewormpairing before and at the beginning of the pairing. Then using suppression subtractive hybrid Technology (suppression subtractive hybridization), SSH) screening of Schistosoma japonicum in male femalewormpairing before (16 days after infection), from early (18 days after infection) and a late (24 days after infection) of the gene difference. By early female cDNA as detection from sub (tester), a pre the female cDNA as driver (driver), sub sub subtractive gene expression detection them enrichment of early gene expression from them, cut detection early and encircle enrichment; encircle late cDNA females For the early detection, a female cDNA as driver, high gene expression detection sub cut them later from enrichment, them are enriched in the early detection of a subtractive gene expression, so as to find the bleeding of female just before and after a period of initial pairing of differentially expressed genes.
Analysis tools for gene encoding all selected proteins were analyzed by GO and KEGG functional classification of metabolic pathways using biological information, according to the analysis results that 28 may be involved in female reproductive development related gene, then RT-qPCR (Real time quantity polymerase chain reaction) in different developmental stages of the female cDNA the template for further validation of the 28 genes.
Separation of ovaries from female schistosome, ovarian total RNA extraction, respectively cDNA and cDNA from adult ovary as template, RT-qPCR was used to observe these reproductive development related genes in ovarian expression.
Results: according to the test of mice infected with different time after the occurrence of a schistosome, determine the mice infected with Schistosoma japonicum 16 days for 18 days to encircle early, just 24 days earlier, a late (basically mature organ). Using SSH technology successfully constructed Japanese blood sucking females from early and early is a the reverse SSH Library, that is, relative to the cDNA gene library of high expression and early pre pairing pairing with respect to the initial stage of high head from gene expression cDNA library. The same method was also constructed from late early and a female schistosome infection, reverse subtractive library, which is relative to the initial stage of high head from gene expression cDNA library and to from late early from high cDNA expression library.
The relative to the early early high expression of a folded gene, relative to the initial stage of high expression from a gene, relative to the high expression of early and late genes from a high relative to the expression of late genes from early pairing sequencing, removing repetitive sequences, sequence, respectively 112, 96, 195 and 172 high gene expression sequence tags (Expressed Sequence Tag, EST), by comparison with the database, get clear annotations were 92,80103 and 90 EST sequences.
Gene Ontology (GO) annotations for the molecular functions of these EST proteins, cell location, and the biological processes involved in these proteins.
Compared with the previous stage to encircle a cyclic compound metabolic process of high expression genes mainly involved in biological processes, metabolism of nitrogenous compounds in the process of cell, macromolecular biosynthesis, redox process.
Compared with the high expression of the early stage of biological processes from pairing genes mainly involved in metabolic processes including nitrogen, phosphorus metabolism, metabolism of cyclic compounds, small molecule metabolism, transport and so on.
From early stage of high relative to encircle expressed genes mainly involved in biological process such as transport, metabolism, carbohydrate metabolism of nucleotides, small molecule metabolism process.
From late early compared to the high expression of biological processes from genes mainly involved in nucleotide metabolism, including transport, phosphorus metabolism, transmembrane transport and so on.
Using the Kyoto Encyclopedia of genes and genomes (Kyoto Encyclopedia of Genes and Genomes, KEGG) on the early high relative to a pairing gene expression relative to the initial stage of high expression of a folded gene, relative to the initial stage of high expression from a base, as well as to the signal pathway of genes encoding proteins involved in a late early expression from high analysis.KEGG analysis found that compared to the previous pairing high expressed genes and early pairing with respect to the high expression of early encircle genes encoding proteins involved in pre pairing 76 signal pathways; from late gene expression relative to a great early stage of high gene expression and relative to a high expression of a late gene encoding this protein from early involvement 79 signal pathways. A number of highly expressed genes involved in the signaling pathway of the main focus To include the metabolic pathway, oxidative phosphorylation, endoplasmic reticulum protein processing, especially the discovery and reproductive and developmental signaling pathways associated gene expression.
Selected to further verify the 28 possible fluorescence quantitative PCR and reproductive development gene related signaling pathways in these signaling pathways, the 18 genes are consistent with the experience of subtractive hybridization. Among the reproduction and development is directly related to the participation of prolactin and estrogen receptor tyrosine kinase signal pathway (tyrosine kinases, Tks), heat shock protein 70 (HSP70 Heat shock protein70) and heat shock protein 90 (Heat shock, proteins90, HSP90), Type V collagen protein TGF- beta signaling pathway, Wnt signaling pathway involved in presenilin proteins, involved in the developmental cycle of yeast cells NIPBL protein (Nipped- beta -like protein) and involved in Hippo signaling pathway the mob protein. The HSP70, HSP90 and presenilin (presenilin) at the gene level from late females was significantly increased, while the Type V tyrosine kinase, collagen protein, NIPBL protein And mob protein in Schistosoma japonicum early gene level females were significantly increased.
RT-qPCR technology on female reproductive organs of female adult ovarian cDNA and cDNA Tyk, HSP90 Type, V collagen protein, presenilin protein, NIPBL protein (Nipped- beta -like protein) and mob protein analysis, the results showed that NIPBL protein and presenilin protein highly expressed in ovary.
Conclusion: This study successfully constructed by suppression of Schistosoma japonicum from early subtractive hybridization from early, late a subtractive cDNA library. Select 7 may directly with the molecular male femalewormpairing of schistosome and oviposition related including Tks, HSP70 and HSP90, Type V, collagen protein, presenilin protein, NIPBL protein, mob protein. Some Tks, HSP70 is involved in schistosome reproductive development has been reported in the literature, Type V, collagen protein, presenilin protein, NIPBL protein, mob protein is involved in schistosome reproductive development function remain to be further clarified.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R532.21

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