大鳞副泥鳅尾鳍再生相关基因PdGig2和PdFrr2的克隆及表达分析

发布时间：2018-01-22 21:25

本文关键词： 大鳞副泥鳅尾鳍再生 Gig2基因 Frr2基因原位杂交　出处：《河南师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：发育成熟的个体在受损后可以在原有组织基础上重建已丢失的组织,这种现象称为再生。所有生物体对组织损伤会做出相应的生理反应,同时与疾病的发生又密切相关,因此研究再生的过程和机制具有重要的意义。哺乳类动物再生能力非常有限,而以蝾螈为代表的有尾两栖类和以斑马鱼为代表的硬骨鱼能够在附肢及鱼鳍截断后通过割处再生完成结构和功能的重建。但两栖类繁殖速度慢,实验室不易饲养,且缺乏相应的遗传操作技术,不能成为理想的研究动物。鱼鳍在切断后却能够完整的恢复原有结构,并且相对简单,易获得,损伤后不危害机体生命,更具意义的是硬骨鱼鱼鳍与人类在内的四足动物肢体早期发育极其相似,但二者再生能力却大不相同,因此鱼鳍可以作为解析分子再生机制的良好体系。本研究先是从已经建立的抑制性消减杂交文库中筛选得到两个鱼鳍再生差异表达明显很大的ESTs,在这两个ESTs的基础上,采用RACE的方法进行扩增,得到两个cDNA全长序列。通过Blast比对后,其中一个基因认为是Gig2基因,另一个Blast结果并未发现有与高度同源的,认为是一个新基因,将其命名为Frr2基因。随后利用DNAMAN,MEGA6.0,sigmaplot等软件对这两个基因进行生物学分析,还测定了这两个基因在不同组织中的表达。Gig2基因是草鱼出血性病毒诱导的基因2,它首先是从紫外失活的GCHV处理的鲫鱼囊胚细胞中鉴定获得的一个新基因。它在鱼类干扰素抗病毒反应中起着重要作用,而在再生中能够调节再生则可能是一个新发现。它cDNA全长是712bp,共编码156个氨基酸,相对分子质量为17.324KDa,等电点(pI)8.69。46个极性氨基酸,71个非极性疏水氨基酸,18个酸性氨基酸,21个碱性氨基酸。蛋白质二级结构发现α-螺旋占23.72%,延伸链占27.56%,β-转角占16.03%,无规卷曲占32.69%。通过半定量以及荧光定量检测发现:在再生后4d表达达到最高水平,在胚胎发育时期的出膜期表达量最高,还发现在精巢组织表达也是最大的。根据Gig2的表达模式我们首次发现了该基因参与到了鱼鳍再生的过程,它可能在鱼鳍结构形成过程中起着调控作用。同源度结果分析发现Gig2基因与其他物种的同源度相对并不是很大,这也使得研究其进化关系引起很大兴趣。Frr2(fin regeneration related gene2)基因是一个未知基因,它是一个与鱼鳍再生相关的一个基因。cDNA全长为1036bp,编码了有88个氨基酸,二级结构发现其中α螺旋占46.59%,β-转角占9.09%,无规卷曲占32.95%,延伸链占11.36%。半定量以及荧光定量检测发现在再生不同时期(0dpa,2dpa,3dpa,4dpa,5dpa,6dpa)的第四天表达最高,在不同组织(肝、心、脑、精巢、肾、卵巢)中心脏表达水平较高,胚胎不同发育时期(囊胚期,原肠胚,神经胚,尾芽期,出膜期)原肠胚期的基因表达水平最高。冰冻切片把处于再生不同时期的尾鳍进行原位杂交。原位杂交结果显示:发现在鱼鳍内部成骨细胞中有表达,在4dpa表达信号最强。我们推测Frr2基因可能参与到了成骨细胞的增殖和分化过程中,与鳍条的形成有关。这两个基因在尾鳍再生过程中表达量发生变化并在再生后期表达量最高,预测在大鳞副泥鳅尾鳍再生过程中起着一定作用,它们可能调控再生过程中鱼鳍结构的形成,而对于他们更多的功能研究仍需进一步的深入研究。
[Abstract]:Mature individuals in the damaged lost tissue can be reconstructed based on the original organization, this phenomenon is called regeneration. All organisms make corresponding physiological response to tissue injury, and disease are closely related, so the research of the regeneration process and mechanism is of great significance. The regeneration ability of mammalian animal is very limited, and as the representative of the salamander amphibians and zebrafish as the representative of the teleost fish in appendages and fins cut by rebuilding the epimorphosis complete structure and function. But the amphibian breeding is slow, laboratories are not easy to raise, and the lack of genetic manipulation of related techniques, can not become a research animal ideal. But after cutting the fins can restore the original structure intact, and relatively simple, easy to obtain, after the injury does not harm the body of life, but more significant is the teleost fin and human The quadruped animal limb early development is very similar, but the two regeneration ability is different, therefore can be used as a good system of fin regeneration mechanism. The analysis of molecular research is established from suppression subtractive hybridization library screened two differentially expressed very obvious fin regeneration of ESTs, based on the two ESTs, by the way of RACE were amplified from two cDNA sequences by Blast. After the comparison, one of the genes that Gig2 gene, another Blast results have not been found and highly homologous, that is a new gene named Frr2 gene. Then the use of DNAMAN. MEGA6.0, SigmaPlot and other biological software analysis of these two genes, also measured the expression of.Gig2 gene of the two genes in different tissues is grass carp hemorrhagic virus induced gene 2, it is first of all from the UV inactivation A new gene was identified GCHV live carp blastula cells in processing. It plays an important role in interferon antiviral response, and in regeneration can regulate regeneration may be a new discovery. Its full length cDNA is 712bp, encoding 156 amino acids, molecular weight was 17.324KDa, isoelectric point (pI) 8.69.46 polar amino acids, 71 non-polar hydrophobic amino acids, 18 amino acids, 21 basic amino acids. Protein two alpha helix structure discovery extension chain accounted for 23.72%, accounting for 27.56%, p-turns accounted for 16.03%, accounting for 32.69%. random coil by semi quantitative and quantitative testing found: the expression of 4D reached the highest level after regeneration, in the period of embryo development hatching the highest expression, also found expression in the testis is also the largest. According to the expression pattern of Gig2 for the first time we found the genes involved in the regeneration of the fin The process, it may play a role in the formation of the fin structure. Gig2 gene was found with other species identity is not large relative analysis of identity results, it also makes research on the evolutionary relationship of.Frr2 (fin regeneration caused great interest in related Gene2) gene is an unknown gene, it is a fin regeneration a related.CDNA gene was 1036bp, encoding 88 amino acids, two level structure found among the alpha helix beta turn accounted for 46.59%, accounting for 9.09%, accounting for 32.95% of random coil, extension chain accounted for semi quantitative 11.36%. and fluorescence quantitative detection in regeneration in different periods (0dpa, 2dpa, 3dpa, 4dpa. 5dpa, 6dpa) on the fourth day of the highest expression in different tissues (liver, heart, brain, kidney, testis, ovary) cardiac expression levels were higher in embryos at different developmental stages (blastula, gastrula, neurula, tail bud stage, hatching) gastrulation base Because the highest expression level. Frozen sections put in different periods of regeneration of the caudal fin in situ hybridization. In situ hybridization results showed that found in osteoblasts with internal fins expression, expression of the strongest signal in 4dpa. We speculated that Frr2 gene may be involved in osteoblast proliferation and differentiation, and fin two. The gene expression changes in regeneration late the highest expression in the caudal fin regeneration process, prediction plays a role in the process of fin regeneration of loach, they may form the regeneration process in the regulation of fin structure, and for their more functional studies still need further research.

【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】：S917.4

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