從五倍的草蝦 fosmid 基因體庫中篩選出抗微生物胜肽的基因

Searching for antimicrobial peptide (AMP) genes from tiger shrimp (Penaeus monodon) using a 5X fosmid genomic library

趙沁梅(Chin-Mei Chao)

指導教授 ： 于宏燦

國立臺灣大學/生命科學院/動物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.10538

摘要

本研究採用四步驟之聚合酵素連鎖反應篩選（4-step PCR screening）的策略，從5倍涵覆率之草蝦 fosmid 基因體庫中，篩選出 AMPs 相關的基因（為 penaeidins 和 crustins）；將找到的殖株定序後、進行基因註解；並和其他物種之同源基因進行比對、加以分類。
篩選結果 ： 找到9個帶有 penaeidin-3 基因5之菌株、3個帶有 penaeidin-5 基因之菌株、6個帶有 crustin(Q1) 基因之菌株、17個帶有 crustin(Q2) 基因之菌株和30個帶有 crustin(Q3) 基因之菌株。估算其在草蝦基因體中存在份數： penaeidin 3 為9份、 penaeidin 5 為3份、 crustin(Q1) 為11份、 crustin(Q2) 為327份和 crustin(Q3) 為204份。
而在 #669-1_10E 之菌株中，我們定序出長1803bp含有標的基因 penaeidin 5之序列，其中辨識出兩個 exons 和一個 intron（長620bp）， open reading frame（ORF）長為79個胺基酸序列。使用演化樹枝分析，結果顯示 penaeidins 可分為四類：penaeidin 2、3、4 和 5。在草蝦中，先前只有 penaeidin 3 之胺基酸序列被辨識出，而 #669-1_10E 之菌株所含之標的基因之胺基酸序列，是與 Fenchi PEN5（第五類只在明蝦中被找到）為同類，在演化樹枝分析結果中與 Fenchi PEN5-1最親近，因此可命名之為 Penmon PEN5-1。
未來，我們亦打算將篩選出之其它標的基因之菌株完成定序。本研究所得之草蝦抗微生物胜肽之序列與其上游調節因子之序列，或許可以提供人工抗微生物胜肽設計之參考，與有用資訊予基因轉殖技術中，將有助於草蝦養殖業之發展。所得之研究結果亦助於研究草蝦之遺傳組成、基因結構等，進而幫助完成草蝦的基因體計畫。

關鍵字

抗微生物胜肽草蝦

並列摘要

Antimicrobial pepitedes (AMP) play a major role in innate immunity, conserved in evolution, and present in all phyla of the living kingdom. They are mostly cationic and amphipathic molecules although they present a great diversity in terms of structural features as well as biological properties and functions. More than 850 antimicrobial peptides have now been discovered in plants, vertebrates, and invertebrates. In shrimp, many kinds of antimicrobial peptides have been characterized, i.e. penaeidins, crustins, crustin-like peptides, single-whey-acidic- domain peptide, anti-lipolysaccharide factors and hemocyanin-derived peptides. Penaeidins were unique in penaeid shrimps and were first characterized from Litopenaeus vannamei. Four peptides (named Pen-2, Pen-3, Pen- 4 and Pen-5) were isolated ,but recent studies just found Pen-3 from Penaeus monodon. Crustin is a widely distributed family and many full-length cDNA or EST had been described in a wide range of decapods, including Panulirs argus, L. vannamei and P. monodon, etc.
We have constructed P. monodon fosmid library of 288000（=750*4*96） independent clones. This clone library represents about 5.3 times coverage （= 1.152*1010/ 2.17*109）of the genome of P. monodon. A simple 4-step PCR screening system was established, and we successfully found clones about genes of penaeidin and crustin. This research offers an opportunity to help achieve genome project of Penaeus monodon.

並列關鍵字

AMPs penaeidin crustin Penaeus monodon fosmid library

參考文獻

Ali MF, Lips KR, Knoop FC, Fritzsch B, Miller C, Conlon JM., 2002. Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata. Biochimicaet Biophysica Acta-proteins and Proteomics 1601 (1): 55-63.

Almeida, R. P., Vanet, A., Witko-Sarsat, V., Meichior, M., McCabe, D. and Gabay, J. E., 1996. Azurocidin, a natural antibiotic from human neutrophils : expression, antimicrobial activity, and secretion. Protein Expr Purif 7,355-366.

Aspán, A., Hall, M. and Söderhäll, K., 1990. The effect of endogenous proteinase inhibitors on the prophenoloxidase activating enzyme, a serine proteinase form crayfish haemocytes. Insect Biochem 20,485-492.

Axen, A., Carlsson, A., Engstrom, A., and Bennich, H., 1997. Gloverin, an antibacterial protein from the immune hemolymph of Hyalophora pupae. Eur J Biochem 247:614-619.

Bartlett TC, Cuthbertson BJ, Shepard EF, Chapman RW, Gross PS, Warr GW., 2002. Crustins, homologues of an 11.5-kDa antibacterial peptide, from two species of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus. marine Biotechnology 4 (3): 278-293.