臺灣博碩士論文加值系統

黃麴毒素(Aflatoxin)為真菌Aspergillus flavus Link及A. parasiticus Speare產生之二次代謝產物，目前已知的種類有B1、B2、G1及G2等四種。黃麴毒素B1合成途徑中的各個步驟，大都被剖析瞭解，至少有22種酵素基因，超過20個酵素步驟參與黃麴毒素B1生合成。這些酵素基因有些已被成功選殖了，本研究乃針對前人由黃麴毒素生合成途徑中之調節基因－aflR (分離自A. flavus)或稱為apa-2 (分離自A. parasiticus)、參與早期生合成之酵素基因－nor-1、avnA、cypX及vbs和晚期生合成之酵素基因－ver-1、omt-1及ord1所設計出之九對引子(primer pairs)，利用PCR的方法，增幅萃取培養於YM培養液中三天之A. flavus、A. parasiticus、A. sojae、A. oryzae及A. niger共20個菌株的DNA，並同時收集濾液，萃取黃麴毒素，藉由薄層色層分析來偵測其是否產毒。由結果顯示，產毒菌株皆具有上述之九個基因，有些非產毒菌株也同時具有上述九個基因。九種生合成相關基因都存在的有A. parasiticus之所有菌株及A. flavus CCRC 30231、CCRC 30166、CCRC 30010，A. oryzae CCRC 30120、 CCRC 30123及A. sojae CCRC 30103等共十五個菌株，經由薄層色層分析，除了A. parasiticus CCRC 30227、A. oryzae CCRC 30120、 CCRC 30123，A. sojae CCRC 30103、A. flavus CCRC 30010這八個菌株是非產毒菌株外，其餘的菌株皆會產毒。但在黃麴毒素生合成基因組成上有缺陷者，一定就是非產毒菌株。產毒菌株與非產毒菌株之間在DNA層次上，藉由PCR的方法偵測其生合成相關基因並無顯著之差異。本研究另外將供試菌株A. parasiticus CCRC 30160、A. parasiticus CCRC 30227及A. flavus CCRC 30231、A. flavus CCRC 30010培養於適合產毒(YES)與不適合產毒(YEP)之培養液中一天，發現A. parasiticus產毒菌株在YES培養液中，vbs、ver-1及ord1會表現；產毒菌株在YEP培養液中，不會有vbs、ver-1及ord1表現。A. flavus產毒菌株在YES培養液中，cypX、omt-1及ord1才表現，產毒菌株在YEP培養液中，無cypX、omt-1及ord1表現。故推測經由RT-PCR的方法，偵測A. parasiticus其vbs、ver-1及ord1和A. flavus其cypX、omt-1及ord1等黃麴毒素生合成相關基因之轉錄差異與產毒有關。在黃麴毒素生合成的調節機制中，不單只受到aflR的調節機制，應是結合生理及環境因子的複合機制。所以可否僅藉由RT-PCR的方法，監控這些基因之轉錄與否來鑑定或區分產毒或非產毒菌株，則仍尚待更進一步之研究證實。

Aflatoxins are the polyketide-derived secondary metabolites. Four aflatoxins, aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) are named from their blue and green fluorescence, respectively. Since 1992, more than 22 genes involved in aflatoxin biosynthesis and over 20 enzymatic steps in the pathway have been cloned and characterized from Aspergillus flavus Link and A. parasiticus Speare. In this study nine previous described pairs of primer encoding target gene and regulatory gene were used for PCR. Among these primers, nor-1、avnA、cypX and vbs encoding early enzymatic conversion in aflatoxin biosynthesis pathway and ver-1、omt-1 and ord1on later steps. In addition, aflR for A. flavus and apa-2 for A. parasiticus are regulatory genes. PCR was used to amplify DNA from mycelia grown in YM broth three days for twenty target fragments from A. flavus、A. parasiticus、A. sojae、A. oryzae and A. niger isolates, respectively. Extracted culture fluid with chloroform and the subsequence TLC analysis can be used to detect aflatoxin production. Aflatoxigenic strains and eight nonaflatoxigenic strains all present the nine genes. PCR has proved to be a very precise and rapid technique for detecting Aspergillus species, but it does not always permit discrimination between aflatoxigenic and some nonaflatoxigenic strains. In other study, we explore aflatoxigenic strains A. parasiticus CCRC 30160, A. flavus CCRC 30231 and nonaflatoxigenic strains A. parasiticus CCRC 30227, A. flavus CCRC 30010 growth in aflatoxin non-permissive YEP broth and aflatoxin-permissive YES broth for one day. Primers encoding nor-1、avnA、cypX、vbs、ver-1、omt-1、ord1 and aflR were used to detecte the gene expression by RT-PCR. TLC analysis showed aflatoxigenic strains grown in aflatoxin permissive YES broth enable produced aflatoxin, while grown in aflatoxin non-permissive YEP broth can not produced aflatoxin. From our study data, vbs、ver-1 and ord1 were expressed on A. parasiticus aflatoxigenic strain (CCRC 30160) when grown in YES broth but not in YEP broth. While A. flavus aflatoxigenic strain (CCRC 30231) grown in YES broth but not YEP broth, cypX、omt-1 and ord1 can transcript. However, the RT-PCR can be used for monitoring aflatoxin production in A. parasiticus according to vbs、ver-1 and ord1expression, as well as cypX、omt-1 and ord1 expression of A. flavus. Aflatoxin biosynthesis gene transcriptional level is not solely dependent on aflR regulatory mechanism. That should conbind environmental and nutritional factors to regulate their gene expression. Consequently, whether aflatoxigenic strains or potential of Aspergillus species being able produced aflatoxin decided from the presence or lack of mRNAs await further characterization.

目錄
頁數
中文摘要---------------------------------------------------------------------------1
英文摘要---------------------------------------------------------------------------3
第一章、 前人研究 -------------------------------------------------------------6
一、 黃麴毒素的性質------------------------------------------------------7
二、 黃麴毒素對人類、動物健康的影響及對經濟的衝擊---------8
三、 產黃麴毒素之真菌菌系--------------------------------------------12
四、 黃麴菌的分類--------------------------------------------------------13
五、 適合黃麴菌產生黃麴毒素的條件--------------------------------15
(一) 溫溼度-------------------------------------------------------------15
(二) 基質----------------------------------------------------------------15
(三) 培養時間----------------------------------------------------------16
六、 黃麴菌之代謝作用與黃麴毒素之生合成---------- ------------17
七、 黃麴毒素對人類、動物健康的影響及對經濟的衝擊--------19
第二章、 黃麴毒素生合成相關基因在黃麴菌產毒與非產毒菌株間組成之差異-------------------------------------------------------------24
前言----------------------------------------------------------------------------25
材料與方法-------------------------------------------------------------------27
一、 菌株來源及保存---------------------------------------------------27
二、 供試菌株總量DNA之萃取-------------------------------------27
(一) 真菌之培養-----------------------------------------------------27
(二) 真菌Genomic DNA之分離----------------------------------27
三、 薄膜色層分析------------------------------------------------------30
四、 參與黃麴毒素生合成酵素基因引子之設計與合成---------31
五、 黃麴毒素生合成相關基因之增幅------------------------------31
(一) PCR反應條件之最適化---------------------------------------31
(二) PCR及multiplex PCR-----------------------------------------31
六、 黃麴毒素生合成相關基因PCR增幅片段之核酸序列分析--------------------------------------------------------------------------32
結果----------------------------------------------------------------------------34
一、 薄膜色層分析------------------------------------------------------34
二、 供試菌株黃麴毒素生合成相關基因之增幅------------------34
三、 黃麴毒素生合成相關基因PCR增幅片段之核酸序列分析--------------------------------------------------------------------------43
討論--------------------------------------------------------------------------49
第三章、黃麴毒素生合成相關基因在黃麴菌產毒與非產毒菌株間表現差異性之探討---------------------------------------------------51
前言---------------------------------------------------------------------------52
材料與方法------------------------------------------------------------------55
一、 供試菌株及培養條件 -------------------------------------------55
二、 總量DNA萃取----------------------------------------------------55
(一) 真菌之培養-----------------------------------------------------55
(二) 真菌genomic DNA分離--------------------------------------55
三、 總量RNA萃取-----------------------------------------------------55
(一) 真菌之培養-------------------------------------------------------55
(二) 全量RNA萃取--------------------------------------------------56
四、 供試菌株培養於不同培養液中之黃麴毒素萃取及薄層色
層分析---------------------------------------------------------------58
五、 供試菌株在不同培養液中生合成相關基因組成差異比較---------------------------------------------------------------------------58
六、 供試菌株在不同培養液中生合成相關基因之表現情形--　59
七、 供試菌株培養於YES與YEP培養液之黃麴毒素萃取及
薄層色層分析------------------------------------------------------59
結果----------------------------------------------------------------------------61
一、 供試菌株在不同培養液之黃麴毒素萃取及薄層色層分析---------------------------------------------------------------------------61
二、 供試菌株培養於YES 培養液中在不同天數下黃麴毒素
產生之情形---------------------------------------------------------61
三、 供試菌株培養於YEP 培養液中不同培養天數下黃麴毒
素產生之情形------------------------------------------------------65
四、 供試菌株在不同培養液中生合成相關基因之PCR增幅--65
五、 利用RT-PCR的方法偵測供試菌株在不同培養液中生合
成相關基因表現情形---------------------------------------------65
討論--------------------------------------------------------------------------75
附錄一----------------------------------------------------------------------------91
附錄二----------------------------------------------------------------------------95
附錄三----------------------------------------------------------------------------96
圖目錄
頁數
第一章
圖一、黃麴毒素B1、B2、G1、G2之結構式。-----------------------------9
圖二、黃麴毒素之生合成路徑。--------------------------------------------21
第二章
圖一、供試菌株培養液萃取所得之黃麴毒素之TLC分析結果。-----35
圖二、供試菌株DNA利用aflR引子之PCR增幅結果。--------------37
圖三、供試菌株DNA利用ord1引子之PCR增幅結果。-------------38
圖四、供試菌株DNA利用avnA引子之PCR增幅結果。-------------39
圖五、供試菌株DNA利用cypX引子之PCR增幅結果。-------------40
圖六、供試菌株DNA利用vbs引子之PCR增幅結果。---------------41
圖七、供試菌株DNA與nor-1、ver-1、omt-1及apa-2等四對引子
進行multiplex PCR之增幅結果。-----------------------------------44
第三章
圖一、利用薄層色層分析的方法偵測供試菌株在YES、YEP、
YM培養液中三天黃麴毒素產生之情形。-----------------------62
圖二、利用薄層色層分析偵測供試菌株在YES培養液培養不同時
間後，黃麴毒素產生之情形。---------------------------------------63
圖三、利用薄層色層分析偵測供試菌株在YES培養液培養不同時
間後，黃麴毒素產生之情形。---------------------------------------64
圖四、利用薄層色層分析偵測供試菌株在YEP培養液培養不同時
間後，黃麴毒素產生之情形。---------------------------------------66
圖五、利用薄層色層分析偵測供試菌株在YEP培養液培養不同時
間後，黃麴毒素產生之情形。---------------------------------------67
圖六、供試菌株於不同培養液中生長後，利用aflR引子增幅之結果。------------------------------------------------------------------------------68
圖七、供試菌株於不同培養液中生長後，利用ord1引子增幅之結果。------------------------------------------------------------------------------69
表目錄
頁數
第一章
表一、黃麴毒素之理化性質---------------------------------------------------10
表二、衛生署公告之食品中黃麴毒素限量標準---------------------------12
第二章
表一、參與黃麴毒素生合成之酵素基因引子----------------------------33
表二、供試菌株產生之黃麴毒素種類-------------------------------------36
表三、供試菌株與黃麴毒素生合成相關基因之PCR結果------------42
表四、供試菌株與黃麴毒素生合成相關基因之Multiplex PCR結果-------------------------------------------------------------------------------45
表五、本試驗供試菌株利用ord1及aflR引子增幅後之產物序列
與NCBI上已登錄之序列比較--------------------------------------47
表六、供試菌株利用aflR及ord1引子增幅後之PCR產物序列比較------------------------------------------------------------------------------48
第三章
表一、Aspergillus parasiticus菌株在YES與YEP培養液中培養一
天黃麴毒素生合成相關基因表現情形----------------------------71
表二、Aspergillus flavus菌株在YES與YEP培養液中培養一天黃
麴毒素生合成相關基因表現情形-----------------------------------72
表三、產毒菌株在YES與YEP培養液中培養一天黃麴毒素生合
成相關基因表現情形--------------------------------------------------74

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