日本三角涡虫Spsb基因的克隆及表达分析

发布时间：2018-02-17 04:11

本文关键词： 日本三角涡虫 Spsb基因再生表达分析　出处：《河南师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：SPSB蛋白家族由四个高度保守的成员(SPSB1-SPSB4)组成,共同特征是其氨基酸包含SPRY结构域和羧基(C)端的SOCS-box基序。虽然在高等动物和一些无脊椎动物中已克隆了Spsb基因并对其表达模式进行了分析,但是,到目前为止,这些基因确切的功能尚不清楚。本研究以日本三角涡虫(Dugesia japonica)为实验材料,克隆了DjSpsb基因的全长cDNA序列并对其表达模式进行了系统分析,结果如下:1、利用RACE技术首次在日本三角涡虫中克隆了Spsb基因的全长c DNA序列。DjSpsb基因的cDNA全长为1251 bp,包含一个105 bp的5′端非翻译区(5′UTR)和一个87 bp的3′端非翻译区(3′UTR)。其中3′端非翻译区包含一个poly(A)尾和一个终止密码子。最大开放阅读框(ORF)为1059 bp,编码一个由352个氨基酸残基组成的蛋白质。2、利用ExPASy,TMHMM 2.0、SMART、Inter Pro等其他在线软件对该基因所推导的氨基酸序列进行生物信息学分析,结果表明:DjSPSB蛋白的分子量为40.90 k Da,理论等电点为9.04,含有一个保守的SPRY结构域和一个SOCS盒,为亲水性核蛋白,存在多个磷酸化位点,无信号肽且不存在跨膜区。3、根据该基因所推导的氨基酸序列利用用贝叶斯MrBayes 3.1.2构建了系统发育树。系统进化分析表明DjSpsb在多细胞动物进化过程中高度保守,且与Spsb1,Spsb2和Spsb4有较高的同源性。4、利用整体原位杂交、切片原位杂交和实时荧光定量PCR技术研究了DjSpsb基因在整体及再生涡虫中的时空表达模式。结果表明:在性未成熟的整体涡虫中,DjSpsb主要在头部表达且边缘部位阳性信号较强,此外,DjSpsb在神经组织和肠支表达;在性成熟的个体中,阳性信号主要在头部、睾丸和卵黄腺部位表达,卵巢部位不表达;在再生个体中,阳性信号在再生胚基表达。RT-PCR结果显示,在尾再生头部分,DjSpsb基因在切割后1,3和5天上调表达且在第5天达到峰值,切割后第7天其表达水平下降且与对照表达水平相似。5、应激反应实验结果显示:热应激和乙醇培养诱导Djspsb基因高表达且在乙醇诱导下呈剂量效应,而高剂量的离子液体抑制其表达。研究结果提示:Djspsb基因可能在涡虫再生过程特别是中枢神经系统形成功能恢复过程中起着重要作用,另外,该基因可能是一个多功能基因,在涡虫生殖系统的发育和应激保护过程中发挥作用。
[Abstract]:The SPSB protein family consists of four highly conserved members, SPSB1-SPSB4. The common feature is that its amino acids contain the SOCS-box motif at the end of SPRY domain and carboxyl motif. Although the Spsb gene has been cloned and analyzed in higher animals and some invertebrates, so far, The exact function of these genes is unclear. In this study, the full-length cDNA sequence of DjSpsb gene was cloned from Dugesia japonica, and its expression pattern was systematically analyzed. The results were as follows: 1: 1. The full-length cDNA sequence of Spsb gene and the cDNA length of Spsb gene were firstly cloned by using RACE technique. The cDNA length of the Spsb gene was 1251 BP, including a 105bp 5'untranslated region (5'UTRR) and an 87bp 3'terminal untranslated region (Utria). The 3'untranslated region contains a polymorphic A) tail and a termination codon. The maximum open reading frame (ORF) is 1059 BP, encoding a protein of 352 amino acid residues. ExPASy-TMHMM 2.0 SMARTInter Pro and other online software were used to detect the gene. The deduced amino acid sequence was analyzed by bioinformatics. The results showed that the molecular weight of the protein was 40.90 kDa, and the theoretical isoelectric point was 9.04. The protein contained a conserved SPRY domain and a SOCS box, which was a hydrophilic nucleoprotein with multiple phosphorylation sites. A phylogenetic tree was constructed by using Bayesian MrBayes 3.1.2 according to the deduced amino acid sequence of the gene. Phylogenetic analysis showed that DjSpsb was highly conserved in the evolution of multicellular animals. It has high homology with Spsb1, SPsb2 and Spsb4. In situ hybridization and real-time fluorescence quantitative PCR techniques were used to study the temporal and spatial expression patterns of DjSpsb gene in the whole and regenerated planaria. The results showed that the expression of DjSpsb gene was mainly in the head and the positive signal was strong at the edge of the whole parasite. In addition, DjSpsb was expressed in nerve tissues and intestinal branches; in sexually mature individuals, the positive signals were mainly expressed in the head, testis and yolk glands, but not in the ovary; in regenerated individuals, the positive signals were expressed in regenerated embryos. The expression of DjSpsb gene was up-regulated on the 3rd and 5th day after cleavage, and reached its peak on the 5th day. On the 7th day after cutting, the expression level of Djspsb decreased and was similar to that of the control. The results of stress reaction showed that heat stress and ethanol culture induced the high expression of Djspsb gene in a dose-dependent manner. The high dose of ionic liquid inhibits its expression. The results suggest that the Djspsb gene may play an important role in the regeneration process, especially in the recovery of central nervous system formation, and it may be a multifunctional gene. Play a role in the development of the reproductive system and stress protection.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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