本文選題：黑斑側褶蛙 + 抗菌肽　； 參考：《東北林業(yè)大學》2016年碩士論文

【摘要】：抗菌肽是兩棲類免疫系統(tǒng)的第一道防線,抗菌肽通常具有較高的多樣性。關于多樣性形成的機制已提出三類機制：抗菌肽基因不斷復制產生新的座位,堿基具有高于其他基因的突變率,并受正選擇的驅動。但是這些過程是如何實現(xiàn)的還不能很好的解釋。我們研究發(fā)現(xiàn),野外環(huán)境下黑斑側褶蛙(Pelophylax nigromaculata)的抗菌肽譜中Temporin-1N表達量最高,且受到強烈的負選擇作用,而其他的抗菌肽則受到不同程度的中性或正選擇作用。我們推測Temporin-1N在整個抗菌譜中起著支柱性作用,為其他抗菌肽減輕了選擇壓力,促進其實現(xiàn)快速進化。為了驗證這一推測,本研究以1只黑斑側褶蛙為材料,通過染色體步移技術解析Temporin-1N抗菌肽的基因及5'-UTR的結構,并對其表達速度及進化機制進行研究。主要結果如下：1. Temporin-1N抗菌肽的基因結構：5'-UTR+編碼信號肽和部分酸性前肽的外顯子1+內含子+編碼剩余酸性前肽和完整成熟肽的外顯子2+3'-UTR。2.一只個體中發(fā)現(xiàn)4個不同的Temporin-1N基因,4個基因的成熟肽編碼區(qū)未發(fā)現(xiàn)堿基突變,顯示極低的遺傳多樣性。3.基于cDNA序列的預測表明,黑斑側褶蛙的Temporin-1N由15個氨基酸殘基組成,為螺旋結構的堿基陽離子肽,與其他物種Temporin理化性質相似,推測其具有廣譜且高效的抗菌活性。4. Neural Network Promoter Prediction預測顯示,Temporin-1N基因5'-UTR和內含子區(qū)均含有多個啟動子區(qū),提示其有快速轉錄的能力。5. TRANSFAC預測顯示,該基因5'-UTR和內含子區(qū)均含有若干個TATA-box、GC-box和CAAT-box真核生物基礎順式作用元件和調控免疫特異性表達及組織特異性表達相關的轉錄因子結合位點,預示Temporin-1N基因可參與多種生理過程。上述結果表明,Temporin-1N具有廣譜的抗菌活性,由多個基因編碼,每個基因具有快速轉錄能力,并參與諸多生理過程,提示Temporin-1N具備在整個抗菌肽譜中起著支柱性作用的條件,預示著正因其庇護,其他抗菌肽才能在輕微選擇壓力下快速積累突變,從而實現(xiàn)快速進化,推動多樣性的形成。
[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.
【學位授予單位】：東北林業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q953

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