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題名 | Differential Gene Expression in Aflatoxigenic Aspergillus Species Containing the Extra Regulatory Gene aflR-2 for Aflatoxin Synthesis in Different Growth Conditions=含有額外調節基因(aflR-2)的產毒素黃麴菌在不同生長條件下其毒素合成基因的表現差異 |
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作者 | 陳瑞祥; 郭榮泰; 黃郁芬; 王婉伶; 蔡竹固; Chen, Ruey-shyang; Kuo, Rong-tai; Huang, Yu-fen; Wang, Wan-ling; Tsay, Jwu-guh; |
期刊 | 稻江學報 |
出版日期 | 20110600 |
卷期 | 5:2 2011.06[民100.06] |
頁次 | 頁51-65 |
分類號 | 369.38 |
語文 | eng |
關鍵詞 | 黃麴毒素; 反轉錄聚合酶連鎖反應; Aflatoxins; aflR1/2; Aspergillus flavus; A. parasiticus; Reverse transcription PCR; RT-PCR; |
中文摘要 | 黃麴毒素為重要食物污染菌Aspergillus flavus及A. parasiticus所產生之二次代謝產物,aflR基因產物是大部份黃麴毒素生合成基因的轉錄活化子。薄層色層(TLC)分析顯示只在A. flavus及A. parasiticus產毒菌株中偵測出黃麴毒素但其它Aspergillus供試菌株則無法偵測出。利用從aflR基因密碼區設計的專一性引子成功地增幅aflR基因之PCR 產物大約1352 bp,PCR 結果顯示在所有產毒菌株及一些非產毒菌株中都能增幅出aflR基因,反之有4個非產毒菌株無法增幅出aflR基因。NcoI限制酵素分析顯示只有A. parasiticus五菌株(71.43%)具有aflR1/2 基因存在。AflR序列比對分析顯示產毒菌株之aflR1/2 基因密碼區與非產毒菌株間有98.5/99.6%相似度,比對aflR1/2基因密碼區序列發現有20至35個核苷酸改變。將供試菌株培養於適合產毒(YES)與不適合產毒(YEP)之培養液中生長一天,藉由反轉錄聚合酶連鎖反應(RT-PCR)偵測黃麴毒素生合成相關基因(nor-1, avnA, cypX, vbs, ver-1, omt-1, ord1及aflR)之轉錄表現,由結果得知A. parasiticus 產毒菌株在YES 培養液中,vbs及ord1會轉錄表現;在YEP培養液中,不會有vbs及ord1轉錄表現。A. flavus產毒菌株在YES培養液中,omt-1會轉錄表現;在YEP培養液中,不會有omt-1轉錄表現。而產毒菌株與非產毒菌株之黃麴毒素產生與aflR1/2、nor-1、avnA、cypX及ver-1 基因是否轉錄表現並無絕對的相關性。aflR持續性表現可能導致代謝路徑中蛋白質的合成,然而與黃麴毒素的產生並無相關性。因此,在能夠評估Aspergillus spp.是否為黃麴毒素產毒菌株之前,黃麴毒素合成的基因調控和aflR的重複數之間的差異尚有待進一步釐清。 |
英文摘要 | Aflatoxins (AF) are polyketide secondary metabolites produced by Aspergillus flavus and A. parasiticus and are important contaminants that often lead to food poisoning. Among Aspergillus species, AF were detected only in aflatoxigenic isolates of A. flavus and A. parasiticus by TLC analysis. AflR gene-specific primers were used to amplify 1352-bp PCR products from all aflatoxigenic and some nonaflatoxigenic isolates. RFLP analysis of the NcoI-digested DNA region of the aflR gene showed that only five of seven A. parasiticus isolates (71.43%) consisted of two aflR genes, aflR1 and aflR2, the nucleotide sequences of which were 98.5/99.6% identical to those of nonaflatoxigenic isolates, demonstrating a change in 20 to 35 nucleotides. To understand the genetic regulation of AF, the tested isolates were grown in AF non-permissive YEP broth and AF-permissive YES broth for one day and reverse transcription PCR (RT-PCR) was used to study the expression of nor-1, aflR, ver-1, avnA, omt-1, ord1, vbs, cypX and β-tublin. Gene expression differed between culture media and species. Vbs and ord1 were expressed in A. parasiticus aflatoxigenic isolates grown in YES, but not YEP, broth, while omt-1 could be transcribed from A. flavus aflatoxigenic isolates grown in YES, but not YEP, broth. Expression of genes aflR1/2, nor-1, avnA, cypX and ver-1 were not correlated with the production of AF. Constitutive expression of aflR could lead to synthesis of pathway proteins, which did not relate to AF accumulation. Therefore, differences in gene regulation and duplication require further characterization in order to assess the benefits for aflatoxigenic Aspergillus species. |
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