筛选日本血吸虫雌虫雌雄合抱前后差异表达基因及功能初步鉴定

发布时间：2018-10-21 14:09

【摘要】：目的:血吸虫病是血吸虫感染引起的一种危害严重的人兽共患病。虫卵不但是血吸虫病的主要传染源也是血吸虫病最重要的致病因子。血吸虫是雌雄异体的吸虫,雌雄合抱的生理学过程对雌虫的性成熟和雌虫产卵的作用是至关重要的。本研究通过基因芯片技术筛选出血吸虫雌雄合抱前后差异表达基因,探究参与雌雄合抱发生的分子机制,寻找出与雌虫性成熟及产卵有关的基因。我们希望这项研究可以为血吸虫病的治疗和预防提供新的靶点。方法:收集血吸虫尾蚴,以1000条/只感染新西兰兔,分别与感染后16天、18天、24天采用灌注法收集虫体,通过预冷PBS和雌雄分离等处理后作为样品送至上海伯豪生物有限公司做基因芯片实验。对芯片结果进行聚类分析筛选出合抱前后差异表达基因,并对这些差异基因进行生物信息学分析。从分析的结果中挑选从合抱初期到合抱后期表达量一直升高的13个基因和表达量下降的2个基因,利用荧光定量PCR验证结果是否与基因芯片一致。结合生物信息学分析结果及荧光定量PCR结果,决定挑选fs800(AY810878.1)、cu-zn-sod(FN316039.1)、eggshell precursor protein(FN317063.1)这三种基因进行下一步功能鉴定的实验。首先通过荧光定量PCR分析上述基因在血吸虫不同发育阶段的表达情况,再利用原位杂交技术对这三种基因进行定位。随后用特异性Sjfs800 si RNA转染28天合抱的血吸虫,并在体外培养10天。通过q PCR在m RNA水平中测量干扰效应。通过激光共聚焦扫描显微镜观察日本血吸虫雌虫卵黄腺的形态学改变并使用光学显微镜观察日本血吸虫雌虫产卵数量的变化。结果:通过SAM对基因芯片筛选的结果进行差异聚类分析,去除重复的基因后,发现合抱后期与合抱前期相比差异表达的基因有198个,合抱后期与合抱初期相比差异表达的基因有132个,合抱初期与合抱前期相比差异表达的基因有26个。用荧光定量PCR对15个差异表达基因进行验证,有13个与基因芯片的结果是一致的,说明利用基因芯片筛选差异表达基因基本可靠。对差异表达基因进行GO分析,结果显示合抱初期相对与合抱前期差异表达基因参与生物学的过程主要集中在氧化还原的过程、含氮化合物的代谢过程及环状化合物代谢过程。合抱后期相对于合抱前期差异表达基因参与生物学过程的基因主要集中在核苷酸代谢过程、碳水化合物代谢过程、磷代谢过程和转运过程。合抱后期相对于合抱初期差异表达基因参与生物学过程主要集中在转运过程、核苷酸代谢过程、碳水化合物代谢过程和磷代谢过程。KEGG分析差异表达基因编码的蛋白参与的代谢通路,结果显示合抱初期相对与合抱前期差异表达基因参与的代谢通路有7条,合抱后期相对与合抱前期差异表达基因参与代谢通路有56条,合抱后期相对与合抱初期差异表达的基因参与的代谢通路有47条。许多基因在TGF-β信号通路,Wnt信号通路、MAPK信号通路、PPAR信号通路中高表达,值得注意的是发现了在生殖发育相关信号通路中存在高表达基因。结合以上实验结果及相关文献的查阅,我们将目光聚集在合抱前期到合抱后期表达量一直升高的基因上,并最终选择了fs800,cu-zn-sod,eggshell precursor protein这三种基因。分析虫卵、尾蚴、16天雌虫、24天雌虫、42天雌虫这五个阶段fs800,cu-zn-sod,eggshell precursor protein的表达情况,其结果显示随着虫体的不断发育,这三者的表达量逐渐升高,均在42天达到高峰,表明这三者与虫体发育分化有着密切关系。利用原位杂交技术分别在28天虫体内对三个基因进行定位。在雌虫靠近卵巢位置的卵黄腺内有fs800的m RNA的阳性信号,eggshell precursor protein的m RNA则主要在雌虫卵模的位置发现有阳性信号,而cu-zn-sod的m RNA阳性信号则在雌雄虫体内均表达。应用Si RNA体外对28天雌雄合抱的成虫进行RNAi,培养10天后,观察发现相对于错配组和空白组,实验组fs800的表达量明显下降,干扰效率达到60%,同时雌雄虫的合抱率和雌虫的产卵量也有一定的下降。激光共聚焦结果显示相对于对照组,干扰组的雌虫的卵黄腺内未成熟的卵黄细胞增多,说明干扰了fs800后对雌虫的卵黄腺的正常发育有一定影响。结论:本实验通过基因芯片技术成功筛选出大量雌虫合抱前后差异表达的基因。通过对这些基因的生物信息学分析,选取了fs800,cu-zn-sod,eggshell precursor protein进行基因定位,发现fs800定位于雌虫的卵黄腺。随后对fs800进行干扰,初步的实验结果显示fs800与雌雄合抱及雌虫产卵有直接的关系,但具体的机制还有待进一步研究。
[Abstract]:Objective: The schistosomiasis is a kind of serious human animal disease caused by schistosome infection. Egg is not but the main source of schistosomiasis is also the most important pathogenic factor of schistosomiasis. Schistosoma japonicum is a paragonimus paragonimus, and the physiological process of male and female clasping plays an important role in the oviposition of female and female worms. This study screened the differentially expressed genes between male and female of Schistosoma japonicum by gene chip technology, explored the molecular mechanism involved in the genesis of male and female, and found genes related to female and female gametophyte and oviposition. We hope that this study will provide new targets for the treatment and prevention of schistosomiasis. Methods: A new Zealand rabbits infected with Schistosoma japonicum were collected by perfusion method on 16 days, 18 days and 24 days after infection, and the samples were sent to Shanghai Bohao Biotech Co., Ltd. to perform gene chip experiment by pre-cooling PBS and separation of male and female. By cluster analysis of chip results, the differentially expressed genes were screened by cluster analysis, and bioinformatic analysis of these genes was carried out. From the results of analysis, 13 genes and two genes whose expression level was decreased from the early stage to the late stage were selected, and whether the results were consistent with the gene chip by fluorescence quantitative PCR. Combined with the results of bioinformatic analysis and fluorescence quantitative PCR, we decided to select the three genes of f800 (AY810878. 1), accession number-zn-sod (FN316039. 1), eggshell precuritidine (FN317063.1). Firstly, the expression of the above genes at different developmental stages of schistosome was analyzed by fluorescence quantitative PCR, and the three genes were located by in situ hybridization technique. Mice were then transfected with specific pf800 si RNA for 28 days, and cultured in vitro for 10 days. Interference effects were measured in m RNA levels by q PCR. The morphological changes of the yellow glands of the female eggs of Schistosoma japonicum were observed by laser confocal scanning microscope and the number of eggs of Schistosoma japonicum was observed with an optical microscope. Results: The results showed that there were 198 genes which were differentially expressed in the late stage and early stage compared with the early stage, 132 of the genes differentially expressed in the late stage and the early stage were found. There were 26 genes differentially expressed in the early stage and early stage. Fifteen differential expression genes were verified by fluorescence quantitative PCR, and the results of 13 genes were consistent with the results of gene chip. The results indicated that the gene chip was used to screen the differentially expressed genes. The expression of differentially expressed genes was analyzed by GO. The results showed that the expression of differentially expressed genes in the early stage was mainly focused on the process of redox, the metabolic process of nitrogen-containing compounds and the metabolic process of cyclic compounds. The gene involved in the biological process is mainly involved in the metabolic process, carbohydrate metabolism, phosphorus metabolism and transport. In the late stage, the differentially expressed genes involved in the initial differential expression of genes involved in the process of transport, metabolic processes, carbohydrate metabolism and phosphorus metabolism. KEGG analyzed the metabolic pathway of protein involved in differentially expressed genes. The results showed that there were 7 metabolic pathways involved in the differentially expressed genes in the early stage of the combination, while 56 were involved in the metabolic pathway with respect to the differentially expressed genes in the late stage. 47 of the metabolic pathways involved in the gene involved in the early phase of the closure compared to the initial differentially expressed genes. Many genes are highly expressed in the signaling pathway, signal transduction pathway, MAPK signal pathway and p38 signaling pathway, and it is worth noting that there are high expression genes in reproductive development-related signaling pathway. In combination with the above experimental results and the reference of relevant literature, we focused our attention on the genes that have been elevated in the early stage to the late stage of fusion, and finally selected the three genes of fs800, p27-zn-sod, eggshell precuricum. The expression of f800, p27-zn-sod and eggshell precuritidine in the five stages of egg, female, 16-day female worm, 24-day female worm and 42-day female worm were analyzed. The results showed that the expression level of these three species increased gradually with the development of pollen tube, all of which reached the peak at 42 days. It is shown that these three are closely related to the development and differentiation of the pituitary gland. Three genes were located in 28-day insect by in situ hybridization technique. The positive signals of the f800 mRNA were found in the yolk glands near the ovary, and the m RNA of the eggshell precuritidine was found to be positive in the position of the female egg mold, while the m RNA positive signal of the n-zn-sod was expressed in both male and female worms. After 10 days of RNAi, the expression level of f800 in experimental group was obviously decreased, and the interference efficiency reached 60%. The results showed that the immature yolk cells in the yolk glands of the female worms of the interfering group were increased with respect to the control group, indicating that the normal development of the yolk glands of the female worm after the f800 was interfered with. Conclusion: The gene chip technology was used successfully to screen the differentially expressed genes of large numbers of female worms. Based on the bioinformatic analysis of these genes, the gene localization was carried out by f800, NCI-zn-sod, eggshell precuricum gene and found that f800 was located in the yolk gland of female worm. The results showed that f800 had a direct relationship with female and female oviposition, but the specific mechanism was still to be studied further.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R383.24

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