黑斑侧褶蛙Temporin-1N抗菌肽基因结构与进化方式的研究

发布时间：2018-05-18 11:17

本文选题：黑斑侧褶蛙 + 抗菌肽　；参考：《东北林业大学》2016年硕士论文

【摘要】：抗菌肽是两栖类免疫系统的第一道防线,抗菌肽通常具有较高的多样性。关于多样性形成的机制已提出三类机制：抗菌肽基因不断复制产生新的座位,碱基具有高于其他基因的突变率,并受正选择的驱动。但是这些过程是如何实现的还不能很好的解释。我们研究发现,野外环境下黑斑侧褶蛙(Pelophylax nigromaculata)的抗菌肽谱中Temporin-1N表达量最高,且受到强烈的负选择作用,而其他的抗菌肽则受到不同程度的中性或正选择作用。我们推测Temporin-1N在整个抗菌谱中起着支柱性作用,为其他抗菌肽减轻了选择压力,促进其实现快速进化。为了验证这一推测,本研究以1只黑斑侧褶蛙为材料,通过染色体步移技术解析Temporin-1N抗菌肽的基因及5'-UTR的结构,并对其表达速度及进化机制进行研究。主要结果如下：1. Temporin-1N抗菌肽的基因结构：5'-UTR+编码信号肽和部分酸性前肽的外显子1+内含子+编码剩余酸性前肽和完整成熟肽的外显子2+3'-UTR。2.一只个体中发现4个不同的Temporin-1N基因,4个基因的成熟肽编码区未发现碱基突变,显示极低的遗传多样性。3.基于cDNA序列的预测表明,黑斑侧褶蛙的Temporin-1N由15个氨基酸残基组成,为螺旋结构的碱基阳离子肽,与其他物种Temporin理化性质相似,推测其具有广谱且高效的抗菌活性。4. Neural Network Promoter Prediction预测显示,Temporin-1N基因5'-UTR和内含子区均含有多个启动子区,提示其有快速转录的能力。5. TRANSFAC预测显示,该基因5'-UTR和内含子区均含有若干个TATA-box、GC-box和CAAT-box真核生物基础顺式作用元件和调控免疫特异性表达及组织特异性表达相关的转录因子结合位点,预示Temporin-1N基因可参与多种生理过程。上述结果表明,Temporin-1N具有广谱的抗菌活性,由多个基因编码,每个基因具有快速转录能力,并参与诸多生理过程,提示Temporin-1N具备在整个抗菌肽谱中起着支柱性作用的条件,预示着正因其庇护,其他抗菌肽才能在轻微选择压力下快速积累突变,从而实现快速进化,推动多样性的形成。
[Abstract]:Antimicrobial peptides are the first line of defense of amphibian immune system. Antimicrobial peptides usually have high diversity. Three mechanisms have been proposed for the formation of diversity: the antimicrobial peptide gene replicates to produce new loci, the base has a higher mutation rate than other genes, and is driven by positive selection. But how these processes are implemented is not well explained. We found that in the field, the Temporin-1N expression of Pelophylax nigromaculata was the highest and strongly negative selection, while the other antimicrobial peptides were neutral or positive selective to varying degrees. We speculate that Temporin-1N plays a columnar role in the whole antimicrobial spectrum, which reduces the selection pressure and promotes the rapid evolution of other antimicrobial peptides. In order to verify this hypothesis, the gene of Temporin-1N antimicrobial peptide and the structure of 5'-UTR were analyzed by chromosome step technique, and the expression rate and evolutionary mechanism of 5'-UTR were studied. The main results are as follows: 1. The gene structure of Temporin-1N antimicrobial peptide encoding signal peptide and exon 1 of partial acidic propeptide encodes exon 23 of the remaining acidic propeptide and the exon 23 of the intact mature peptide. Four different Temporin-1N genes were found in one individual, and no base mutation was found in the mature peptide coding region of 4 genes, indicating very low genetic diversity. The prediction based on cDNA sequence showed that the Temporin-1N of Rana nigra was composed of 15 amino acid residues and was a base cationic peptide with helical structure, which was similar to the physical and chemical properties of other species Temporin, and its antibacterial activity was presumed to be broad spectrum and high efficiency. 4. Neural Network Promoter Prediction prediction showed that both the 5'-UTR and intron regions of the gene had multiple promoter regions, indicating that they had the ability of rapid transcription. TRANSFAC prediction showed that both the 5'-UTR and intron regions of the gene contained several basic cis-acting elements of TATA-boxbox and CAAT-box eukaryotes and transcription factor binding sites related to the regulation of immuno-specific expression and tissue specific expression. It is suggested that Temporin-1N gene may be involved in many physiological processes. These results suggest that Temporin-1N has broad-spectrum antibacterial activity and is encoded by multiple genes. Each gene has a rapid transcription ability and participates in many physiological processes, suggesting that Temporin-1N has the condition of playing a columnar role in the whole antimicrobial peptide spectrum. It is indicated that other antimicrobial peptides can rapidly accumulate mutations under mild selection pressure and thus achieve rapid evolution and promote the formation of diversity.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q953

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