臺灣博碩士論文加值系統

Aspergillus oryzae是醱酵工業上經常使用的菌種。此種真菌能分泌多種蛋白酉每，以分解環境中的物質，作為營養來源。Leucine aminopeptidase (LAP) 可能也是扮演相同的角色。在市面上已經有來自A. oryzae 的LAP商品，但是目前仍未有lap基因序列及其生理意義的相關文獻被發表。
為取得lap基因片斷做為探針，將市售的Aspergillus sojae蛋白 酉每，經FPLC及RP-HPLC純化出LAP，再分別以endopeptidase Glu-C及lysylendopeptidase處理後，回收純化胜月太。經氨基酸定序分析，將定序所得的氨基酸序列，設計成degenerate primer，以A. oryzae cDNA為模板 (template)，利用聚合酉每 鏈鎖反應 (PCR)，複製出700 bp的片段。以此片段作為探針 (probe)，由A. oryzae染色體基因庫及cDNA基因庫中，分別選殖出lap基因。
A. oryzae lap基因的ORF全長為1,320 bp，帶有3個introns，可轉譯出377個氨基酸(40,506 Da)。轉譯出的氨基酸序列，與V. proteolyticus lap基因具有33 % 的相似性。將A. sojae LAP蛋白之N端氨酸基序列與A. oryzae lap基因比對，顯示在A. oryzae lap基因中，具有由77個氨基酸組成的訊號胜月太 (signal peptide)，俾將LAP外泌到胞外。分泌到胞外的LAP，分子量為32,680 Da，與利用SDS-PAGE上所測得的分子量 (35 kDa) 接近。在距ATG -120 ~ -114，有一TATA box，另有CAAT box位於-286 ~ -283。在-88 ~ -69位置，有一個特殊的DNA序列，可能是pH的調控區域。在ORF的3''端，有poly (A) adding site。在ORF內的3個introns，大小分別為59、74及56 bp，距離ATG 155-213、640-713 及1031-1086的位置。除了最靠近轉錄起始點的intron外，其餘兩個introns的結構，皆符合GT-AG的原則。lap基因的Asn-87與Asn-288，可能是會被醣化的位置。

Aspergillus oryzae is widely used in the fermentation industry. This fungus can to secrete many types and high level of proteases to decompose nutrient sources. Leucine aminopeptidase (LAP) may participate in protein degradation. A. oryzae LAP is commercialized, however, its gene structure and physiological role has never been reported.
To obtain a lap gene fragment as probe, LAP was purified from commercial Aspergillus sojae proteases using FPLC and RP-HPLC. The purified LAP was digested with endopeptidase Glu-C or lyslyendopeptidase. Partial LAP fragments were sequenced and back-translated to degenerate primers. A 700bp fragment was amplified by PCR using A. oryzae cDNA as template. The lap genes were then isolated from A. oryzae genomic and cDNA libraries using 700 bp fagment as probe.
A. oryzae lap gene comprises 1,320 bp with 3 introns and encodes a protein consisting of 377 amino acids ( 40,506 Da). The deduced amino acids shows only 33% identity to V. proteolyticus lap. Comparison of the protein sequencing from A. sojae LAP and the deduced amino acid sequence from A. oryzae lap revealed a signal peptide with 77 amino acids in A. oryzae LAP. The mature protein has a calculated molecular mass of 36,280 Da which almost matches the molecular mass of 35 kDa determined from SDS-PAGE. A TATA box was identified at position -120 from the ATG, and a CAAT box was found at position -286. A specific region, -88 to -69, may be related to pH regulation. A poly (A) adding site was found in the 3'' region. Three introns are 59, 74, and 56 bp in size and located at 155-213, 640-713 and 1031-1086, respectively. Except the intron which is the nearest to start codon, others obeys the GT-AG rule. Two putative N-glycosylation sites could be identified in Asn-87 and Asn-288 of A. oryzae lap.

Abstract ---------------------------------------------------------------------1
中文摘要 -------------------------------------------------------------------3
前言 -------------------------------------------------------------------------5
材料與方法
一、藥品 -----------------------------------------------------------------12
二、菌株與質體----------------------------------------------------------12
三、E. coli質體DNA的小量抽取-----------------------------------13
四、DNA的洋菜電泳分析 -------------------------------------------14
五、DNA片段的回收及純化
(1).回收小片段DNA --------------------------------------------------15
(2).回收大片段DNA --------------------------------------------------16
六、DNA及RNA純鑑定及定量分析 -----------------------------16
七、引子的設計 --------------------------------------------------------17
八、聚合酵素鏈鎖反應 -----------------------------------------------18
九、反轉錄聚合酉每鍊鎖反應 -----------------------------------------19
十、DNA黏接反應 ----------------------------------------------------20
十一、質體的轉形作用
(1).勝任細胞的製備 --------------------------------------------------21
(2).轉形作用 -----------------------------------------------------------21
十二、蛋白質電泳分析 -----------------------------------------------22
十三、Leucine aminopeptidase (LAP)活性測定
(1).酵素呈色法 ---------------------------------------------------------24
(2).LAP活性染色-------------------------------------------------------24
十四、DNA探針的取得
(1).LAP的純化 --------------------------------------------------------25
(2).反相高效液相層析法 --------------------------------------------26
(3).以protease切LAP ------------------------------------------------27
(4).液相層析-三重四極質譜儀 -------------------------------------29
(5).蛋白的定序 ---------------------------------------------------------29
十五、Aspergillus oryzae染色體DNA的抽取 -------------------30
十六、A. oryzae RNA的萃取 ----------------------------------------31
十七、mRNA的純化 --------------------------------------------------33
十八、A. oryzae基因庫的建立
(1).染色體基因庫的建立 --------------------------------------------34
(2).cDNA基因庫的建立 --------------------------------------------- (3).基因庫的放大 ------------------------------------------------------3539
(4).含phagemid之噬菌體的大量釋出法 -------------------------40
(5).探針的製備 ---------------------------------------------------------41
(6).溶菌斑雜交法 ------------------------------------------------------42
(7).單一選殖株質體釋法 --------------------------------------------43
(8).含lap基因之DNA片段的限制酉每圖譜 ----------------------45
(9).南方轉漬雜交法 ---------------------------------------------------45
十九、質體pBK-CMV1的次選殖 ----------------------------------46
二十、蛋白含醣量之分析 --------------------------------------------46
二十一、A. oryzae培養液的濃縮 -----------------------------------47
結果
一、lap基因探針的製備 ----------------------------------------------48
(一).根據已知LAP酵素氨基酸序列的保留區 ------------------48
(二).根據Aspergillus sp. LAP酵素的氨基酸序列 --------------48
二、A. oryzae染色體基因庫及cDNA基因庫的構築 -----------52
三、lap基因的篩選 ----------------------------------------------------53
四、含有lap基因之DNA片段之限制酉每圖譜分析 -------------54
五、pBK-CMV1及pSK3的核酸定序 -----------------------------55
六、lap基因的結構 ----------------------------------------------------56
討論 -------------------------------------------------------------------------57
圖表 -------------------------------------------------------------------------63
參考文獻 -------------------------------------------------------------------103

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