日本三角涡虫热休克蛋白70和90家族相关基因表达及功能分析
发布时间：2018-09-01 06:54
【摘要】：日本三角涡虫(Dugesia japonica)隶属扁形动物门(Platyhelminthes),涡虫纲(Turbellaria),在动物系统演化中占有重要地位,并且具有极强的再生能力。目前,虽然已克隆大量与涡虫再生相关的基因,但其具体的再生机制及相关基因的功能仍不清楚。本文从日本三角涡虫转录组数据库中筛选出再生过程中持续上调表达但表达量较低的热休克蛋白70和90家族的Djhsp70e、Djhsp90a、Djhsp90b和Djhsp90c基因。利用整体原位杂交、RNA干扰和Real time-PCR等分子生物学技术对四基因的时空表达模式和功能进行分析,结果如下:(1)通过Real time-PCR技术对切割损伤、离子液体暴露、冷应激和热应激环境下日本三角涡虫体内四基因的表达量进行分析,结果显示:在应激条件下,四基因的表达量均明显上调,并且随着切割损伤时间的延长、离子液体浓度的升高,四基因的表达量逐渐上升。(2)日本三角涡虫Djhsp70e基因的cDNA长度为1982bp,5’端有180bp非编码区,3’端有38bp的非翻译区,其中包含一个长度为1764bp的最大开放阅读框(ORF),编码一个由587个氨基酸构成的蛋白质,含有两个热休克蛋白70家族所特有的标签序列。原位杂交结果显示:在整体中,该基因在除脑及咽部以外的身体各部分广泛表达,身体两侧和肠支处杂交信号较强;再生个体的芽基部位有较强的杂交信号。RNA干扰该基因后,整体涡虫出现严重头部溶解且不能再生,而头再生尾片段也出现严重的溶解现象。Real time-PCR结果表明:干扰该基因后,Djpiwi-1和Djpiwi-b的表达量明显下调。(3)日本三角涡虫Djhsp90a基因cDNA长为3212bp,包括5’端1039bp的非编码区和3’端22bp的非编码区,其中包含一个长度为2151bp的ORF,编码一个由716个氨基酸构成的蛋白质。其中包含6个热休克蛋白90家族所特有的序列标签。原位杂交结果显示:在整体涡虫中杂交信号主要分布在除脑及咽以外的大部分实质组织中;在再生个体中的芽基部位有较强杂交信号。RNA干扰该基因后,整体涡虫出现明显的头部溶解现象并伴有身体持续性皱缩;头再生尾片段再生被抑制;尾再生头片段与对照比没有明显变化。Real time-PCR结果表明:干扰该基因后,Djpiwi-1和Djpiwi-b的表达量明显下调。(4)日本三角涡虫Djhsp90b基因的cDNA长度为2636bp,5’端有94bp的非编码区和3’端有132bp非编码区,含有2409bp的最大开放阅读框,编码一个由802个氨基酸构成的蛋白质。该基因所推导的氨基酸序列含有5个HSP90家族所特有的标签序列。整体原位杂交结果显示:该基因在整体涡虫实质组织中广泛表达且身体两侧有较强的杂交信号;再生个体的芽基部位杂交信号较强。RNA干扰该基因后,整体涡虫身体出现明显的皱缩;头再生尾和尾再生头片段再生速度缓慢。Real time-PCR结果表明:干扰该基因后,Djpiwi-1和Djpiwi-b的表达量明显下调。(5)日本三角涡虫中Djhsp90c基因的cDNA长为2573bp,其中包含一个长度为2394bp的最大开放阅读框,编码一个由797bp氨基酸编码的蛋白质,5’端有114bp的非编码区,3’端有65bp的非编码区。包含6个HSP90家族基因所特有的标签序列。原位杂交结果显示:该基因在整体涡虫中杂交信号主要分布在身体两侧及尾部;再生片段芽基部位有较强的杂交信号。RNA干扰该基因后,整体涡虫口部两侧出现膨胀现象;头再生尾和尾再生头片段再生受到抑制。Real time-PCR结果表明干扰该基因后,Djpiwi-1、Djpiwi-b和Djh2b基因的表达量显著下调。以上结果表明:涡虫体内Djhsp70e、Djhsp90a、Djhsp90b和Djhsp90c基因在应激条件均具有细胞保护作用;四基因通过调控干细胞维持涡虫体内组织平衡,并在再生过程中起重要作用。除此之外,Djhsp90c基因通过调节Djpiwi-b和Djh2b基因调节涡虫体内干细胞的增殖和分化,是涡虫再生过程中重要的调节因子。
[Abstract]:Dugesia japonica belongs to the phylum Platyhelminthes and Turbellaria. It plays an important role in the evolution of animal system and has strong regeneration ability. In this paper, Djhsp70e, Djhsp90a, Djhsp90b and Djhsp90c genes, which were continuously up-regulated but low-expressed during regeneration, were screened from the transcriptome database of Trichomonas japonica. Temporal and spatial expression patterns and functions of the four genes were studied by in situ hybridization, RNA interference and Real time-PCR. The results were as follows: (1) Real time-PCR was used to analyze the expression of four genes in Trichomonas japonica under cutting injury, exposure to ionic liquids, cold and heat stress. The results showed that the expression of the four genes was significantly up-regulated under stress conditions, and ionic liquids increased with the prolongation of cutting injury time. (2) The length of the Djhsp70e gene was 1982 bp, the non-coding region was 180 BP at the 5'end and the untranslated region was 38 BP at the 3'end, which contained the largest open reading frame (ORF) with a length of 1764 BP encoding a protein of 587 amino acids and two heat breaks. The results of in situ hybridization showed that the gene was widely expressed in all parts of the body except the brain and pharynx, and the hybridization signals were strong in both sides of the body and in the intestinal branches; the hybridization signals were strong in the bud base of the regenerated individuals. The expression of Djpiwi-1 and Djpiwi-b was significantly down-regulated after interfering with the gene. (3) The length of the Djpiwi-1 and Djpiwi-b gene was 3212 bp, including the non-coding region of 5'end 1039 BP and the non-coding region of 3'end 22bp. In situ hybridization showed that hybridization signals were mainly distributed in most parenchymal tissues except brain and pharynx, and strong hybridization signals were found in the bud base of regenerated individuals. The expression of Djpiwi-1 and Djpiwi-b in T. japonicum was significantly down-regulated after RNA interference with the gene. The length of the B gene cDNA is 2636 bp, the 5'end has 94 BP non-coding region and the 3'end has 132 BP non-coding region. It contains the largest open reading frame of 2409 bp, encoding a protein composed of 802 amino acids. The amino acid sequence derived from the gene contains five tag sequences unique to the HSP90 family. The results of Real-time-PCR showed that Djpiwi-1 and Djpiwi-1 were interfered with the gene. The expression of jpiwi-b was significantly down-regulated. (5) The length of the Djhsp90c gene was 2573 bp, which contained a 2,394 BP open reading frame encoding a protein encoded by 797 BP amino acids, 114 BP non-coding region at the 5'end and 65 BP non-coding region at the 3'end. It contained six HSP90 family genes. The results of in situ hybridization showed that the hybridization signals of the gene were mainly distributed on both sides of the body and tail of the whole worm, and strong hybridization signals were found in the bud base of regenerated fragment. The results showed that Djpiwi-1, Djpiwi-b and Djh2b genes were significantly down-regulated after the gene was interfered with. In addition, Djhsp90c gene regulates the proliferation and differentiation of stem cells in vivo by regulating Djpiwi-b and Djh2b genes, and is an important regulator in the process of regeneration.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
本文编号：2216449
[Abstract]:Dugesia japonica belongs to the phylum Platyhelminthes and Turbellaria. It plays an important role in the evolution of animal system and has strong regeneration ability. In this paper, Djhsp70e, Djhsp90a, Djhsp90b and Djhsp90c genes, which were continuously up-regulated but low-expressed during regeneration, were screened from the transcriptome database of Trichomonas japonica. Temporal and spatial expression patterns and functions of the four genes were studied by in situ hybridization, RNA interference and Real time-PCR. The results were as follows: (1) Real time-PCR was used to analyze the expression of four genes in Trichomonas japonica under cutting injury, exposure to ionic liquids, cold and heat stress. The results showed that the expression of the four genes was significantly up-regulated under stress conditions, and ionic liquids increased with the prolongation of cutting injury time. (2) The length of the Djhsp70e gene was 1982 bp, the non-coding region was 180 BP at the 5'end and the untranslated region was 38 BP at the 3'end, which contained the largest open reading frame (ORF) with a length of 1764 BP encoding a protein of 587 amino acids and two heat breaks. The results of in situ hybridization showed that the gene was widely expressed in all parts of the body except the brain and pharynx, and the hybridization signals were strong in both sides of the body and in the intestinal branches; the hybridization signals were strong in the bud base of the regenerated individuals. The expression of Djpiwi-1 and Djpiwi-b was significantly down-regulated after interfering with the gene. (3) The length of the Djpiwi-1 and Djpiwi-b gene was 3212 bp, including the non-coding region of 5'end 1039 BP and the non-coding region of 3'end 22bp. In situ hybridization showed that hybridization signals were mainly distributed in most parenchymal tissues except brain and pharynx, and strong hybridization signals were found in the bud base of regenerated individuals. The expression of Djpiwi-1 and Djpiwi-b in T. japonicum was significantly down-regulated after RNA interference with the gene. The length of the B gene cDNA is 2636 bp, the 5'end has 94 BP non-coding region and the 3'end has 132 BP non-coding region. It contains the largest open reading frame of 2409 bp, encoding a protein composed of 802 amino acids. The amino acid sequence derived from the gene contains five tag sequences unique to the HSP90 family. The results of Real-time-PCR showed that Djpiwi-1 and Djpiwi-1 were interfered with the gene. The expression of jpiwi-b was significantly down-regulated. (5) The length of the Djhsp90c gene was 2573 bp, which contained a 2,394 BP open reading frame encoding a protein encoded by 797 BP amino acids, 114 BP non-coding region at the 5'end and 65 BP non-coding region at the 3'end. It contained six HSP90 family genes. The results of in situ hybridization showed that the hybridization signals of the gene were mainly distributed on both sides of the body and tail of the whole worm, and strong hybridization signals were found in the bud base of regenerated fragment. The results showed that Djpiwi-1, Djpiwi-b and Djh2b genes were significantly down-regulated after the gene was interfered with. In addition, Djhsp90c gene regulates the proliferation and differentiation of stem cells in vivo by regulating Djpiwi-b and Djh2b genes, and is an important regulator in the process of regeneration.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
【参考文献】
相关博士学位论文 前2条
1 程方方;日本三角涡虫Neurotrophin信号通路关键基因及flotillins基因的克隆及功能分析[D];河南师范大学;2016年
2 马克学;持久饥饿对日本三角涡虫细胞形态结构、生化代谢和基因表达的影响[D];河南师范大学;2014年
相关硕士学位论文 前1条
1 陈静;日本三角涡虫Spsb基因的克隆及表达分析[D];河南师范大学;2016年
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