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標題: Steinernema abbasi蟲生線蟲之Xenorhabdus indica共生菌的鑑定及其代謝物與質體之分析
Identification, and analyses of metabolites and plasmids in the symbiotic bacterium, Xenorhabdus indica, of the entomopathogenic nematode, Steinernema abbasi
作者: 蔡米皓
Tsai, Mi-Hau
關鍵字: 蟲生線蟲
Entomopathogenic nematode
Steinernema abbasi
Xenorhabdus indica
Steinernema abbasi
symbiotic bacterium
Xenorhabdus indica
Galleria mellonella
cell line
insecticidal and antimicrobial substances
出版社: 昆蟲學系所
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摘要: 台灣產之蟲生線蟲,Steinernema abbasi,於1998年在花蓮縣甘藷田中採集土壤,經斜紋夜蛾(Spodoptera litura)幼蟲誘釣而得,其腸道內所含之共生菌經細胞型態、培養特性、生理生化特性,測定結果應屬於Xenorhabdus屬的共生菌;在16S rDNA部分序列所建構之系統發生樹,顯示台灣產之蟲生線蟲共生菌種應鑑定為Xenorhabdus indica。共生菌在體外培養下僅原生型菌 (primary form) 可分泌具有殺蟲活性之代謝物,對昆蟲細胞株Sf21及S2會造成細胞壞死 (necrosis),但對仔倉鼠腎細胞株 BHK-21則無活性。共生菌濾液在體外處理大蠟蛾 (Galleria mellonella) 及哺乳動物血球,於24 h後之壞死率,顯示濾液對大蠟蛾血球具破壞性,對紅血球則無溶血現象。去活性之原生型共生菌體對大蠟蛾幼蟲會造成嚴重麻痺進而死亡,此病徵與萃取原生型菌之脂多醣體 (lipopolysaccharide, LPS) 注入大蠟蛾幼蟲所產生的症狀相似,顯示X. indica之LPS對大蠟蛾是具神經毒。去活性之原生型共生菌體對哺乳動物及昆蟲血球均會造成溶血及壞死現象。原生型共生菌之LPS對大蠟蛾血球無破壞性,顯示LPS並非以破壞血球之免疫系統為主要因素。由以上結果推論,共生菌釋放到昆蟲血體腔中所分泌之LPS及某些代謝物,會抑制昆蟲免疫系統,以致共生菌及線蟲能順利增殖,並引起敗血症,在短時間內殺死昆蟲。共生菌X. indica在體外培養72 h後,在此生長期可產生殺蟲物質及廣效性抗菌物質,對9種人體病原細菌及植物病原真菌具有抑制性。濾液經不同孔徑分子篩萃取後,具有抗生活性之物質主要分佈在100及10 kDa分子篩中;而對昆蟲Sf21細胞株造成細胞壞死,主要在10及3 kDa。殺蟲物質之生物檢定結果,僅10 kDa者具殺蟲性,於注入血腔後24 h對大蠟蛾之幼蟲死亡率達96.67%,顯示10 kDa所篩之物質兼具抑菌及殺蟲性。濾液萃取物之蛋白質電泳分析,發現100 kDa分子篩物質分子量約為85 kDa;而10 kDa分子篩之萃取物,其分子量約為22及25 kDa。經蛋白質之呈色反應結果,不同分子篩之篩出物均屬於蛋白質或胜肽之結構。在不同孔徑分子篩中萃取之濾液,50-10 kDa範圍中可測得exo及endo兩型幾丁質分解酵素。由共生菌所純化之LPS濃度約3×105 EU/ml,於36 h後造成大蠟蛾幼蟲死亡率達93%。經測試結果發現X. indica 之LPS對Bacillus subtilis及Botrytis cinerea均可引起約7.67 mm之抑制圈;而Xenorhabdus nematophila約為8.00與5.33 mm,但同為腸內菌科之Escherichia coli,其LPS對B. subtilis之抑制圈僅約1.33 mm,顯示X. indica之LPS對細菌生長及真菌孢子發芽具有抑制作用。本蟲生線蟲之共生菌 (X. indica) 含有環型結構之質體,其分子大小為5,167 bp,並發現其GC=39%,AT=61%,故屬於AT rich的DNA序列。解序後只有7個胺基酸序列具有相似之功能基因。質體之基因推測與細胞膜之構成、致病之溶血素、醣代謝及孢子發芽等相關基因所合成之胺基酸序列有關,但仍然有許多未知功能基因,尚待進一步研究。
The symbiotic bacterium of the entomopathogenic nematode, Steinernema abbasi, isolated from Taiwan, was determined to be the genus Xenorhabdus based on physiological and biochemical characteristics. It was further identified to be similar to Xenorhabdus indica of S. abbasi Oman isolate as identified by the sequence analyses of its 16S rDNA. The cultured filtrates of Xenorhabdus indica from only the primary form were toxic to Sf21 and S2 cell lines, while those of both forms were not toxic to a mammalian cell line. The necrotic rates of Galleria mellonella hemocytes at 24 h after treating with the cultured filtrates of symbiotic bacteria were significantly different from those of the control, whereas the rates treated with X. indica lipopolysaccharide (LPS) were similar to those of the control. These results indicate that necrosis in G. mellonella hemocytes occurs at 24 h after treatment with the filtrates, while erythrocytes as treated with filtrates were not significantly different, revealing that the culture filtrates do not contain hemolytic substances. Inactivated bacterial cells (primary form) caused serious paralysis in G. mellonela larvae and eventually killed insects. This symptom was found to be similar to that injected with LPS extracted from the primary form. Therefore, it is suggested that LPS is neurotoxic to G. mellonella larvae. Both hemolytic rates of mammalian and insect hemocytes treated with inactivated bacteria (primary form) were capable of causing necrosis. In in vivo assays, the inactivated bacterial cells were capable of causing necrosis and subsequently killed hemocytes of both G. mellonella and Spodoptera litura larvae; however, they were comparatively lesser destructive to S. litura hemocytes. In in vitro assays, LPS from X. indica (primary form) was not markedly detrimental to G. mellonella hemocytes compared with the control groups, suggesting that LPS is not a major factor affecting insect immune system. Therefore, it is speculated that LPS and certain substances when released into insect hemocoel from symbiotic bacteria could hamper insect immune system, resulting in proliferation of symbiotic bacteria and nematodes, and subsequently causing septicemia to rapidly kill their insect hosts. Xenorhabdus indica caused ca. 95% mortality of Galleria mellonella mature larvae at 72 h after culturing, indicating that this bacterium secreted insecticidal substances in its culture medium. The cultured filtrates could also inhibit nine kinds of human pathogens and plant pathogenic fungi. The cultured filtrates screened through 10 or 100 kDa molecular sieves could inhibit the growth of Bacillus subtilis and Botrytis cinerea while those through 3-kDa sieve could inhibit B. subtilis only. However, only the filtrates through 10-kDa sieve resulted in 96.67% mortality of G. mellonella larvae at 24 h. It is thus indicated that both insecticidal and antimicrobial substances are present in the 10-kDa sieved filtrates. Proteins in the cultured filtrates were analyzed using SDS-PAGE electrophoresis. A protein band with 85 kDa of molecular weight was detected in the 100-kDa sieved filtrates while two bands with 22 and 25 kDa were found in the 10-kDa sieved filtrates. On the basis of coloration tests, most of the separated molecules showed amino acid structures. Furthermore, both exo- and endo-chitinases in the filtrates through 10-50 kDa sieves could be detected after reacting with different substrates, emitting fluorescence under the UV microscope. The concentration of LPS isolated from X. indica was ca. 3 x 105 EU/ml, causing ca. 93% mortality of G. mellonella larvae at 36 h, respectively. The LPS from X. indica resulted in ca. 7.67 mm of inhibition zone against a bacterium, B. subtilis and a fungus, B. cinerea, whereas that from Xenorhabdus nematophila caused ca. 8.00 and 5.33 mm of inhibition zone, respectively. In contrast, LPS from Escherichia coli which is also an intestinal bacterium produced only ca. 1.33 mm of inhibition zone against B. subtilis. Therefore, the LPS from X. indica could inhibit both bacterial and fungal growth. Electron micrographs showed a circular form of DNA structure in X. indica plasmids, its size being ca. 5,167 bp, with GC=39%, AT=61%, indicating an AT-rich DNA sequence. As a result from BLAST analysis, only 7 fragments contained similarly functional genes. It was also speculated that X. indica plasmids seem to be involved in cellular membrane formation, pathogenic hemolysin, glucose metabolism, spore germination, and others. However, further studies on many other unknown functional genes which are related to syntheses of amino acid sequence leading to these functions remain to be undertaken.
其他識別: U0005-2706201215515500
Appears in Collections:昆蟲學系



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