結球萵苣為菊科萵苣屬一年生草本植物，為臺灣重要之外銷作物。結球萵苣於採收後其傷口處會逐漸轉變為紅褐色，此情形會降低商品之外觀價值，並使消費者之購買意願下降。褐變之生成與酚類物質受到多酚氧化酶(polyphenol oxidase, PPO)催化，進而導致氧化相關。目前已知創傷會誘導結球萵苣主莖切片苯丙胺酸氨基裂解酶(phenylalanine ammonia lyase, PAL)活性提升，使酚類物質含量上升，此現象被視為結球萵苣主莖褐變之關鍵控制點，本研究欲從基因層次探討PAL同源基因表現量與萵苣切面褐變之關係。

利用萵苣公開之基因體資料庫，共找出六條具有PAL酵素基因註解(gene annotation)之序列，以專一性之引子進行全長PCR放大，成功以gDNA擴增出Lsa002790.1與Lsa019010.1；以cDNA放大出Lsa005270.1、Lsa027790.1、Lsa019025.1之全長片段。並以其與已發表之LsPAL1間的相似程度，將其依序命名為LsPAL1至LsPAL5，其中LsPAL5具兩個不同起始子之亞型。半定量PCR結果顯示創傷後2小時內LsPAL1、LsPAL2、LsPAL3、LsPAL4表現量皆受創傷誘導上升，於創傷後8小時前後具有最高之表現量。然而，LsPAL5於創傷前後基因皆未有表現。推測LsPAL基因家族中LsPAL1、LsPAL2、LsPAL3、LsPAL4與萵苣切面褐變具有關聯性，而LsPAL5則與褐變產生無直接關係。

由前人研究得知數種可抑制結球萵苣切面褐化之藥劑處理，本研究將結合此些藥劑處理，藉其對於抑制褐變之效果，判斷各褐變抑制劑於酵素型褐變中所扮演之角色，並藉此瞭解LsPAL各同源基因與主莖切面褐變之關係。浸泡3%醋酸5秒鐘可使主莖切片維持原色澤至處理後10天；3%正丁醇處理5分鐘雖能延後褐變上升之時間點，但於處理後10天仍出現褐變現象；而半胱胺酸鹽酸鹽(L-cysteine hydrochloride, CysH)處理5分鐘亦能抑制褐變發生，唯處理後10天部分樣本顏色轉為黃綠色。酵素活性部分，醋酸能有效抑制PAL活性於處理後72小時內不上升；正丁醇和CysH處理則分別延緩PAL活性至處理後72小時與處理後48小時才上升。此外，醋酸與正丁醇處理皆使PPO活性上升，CysH處理則使PPO活性於處理後2天內萵苣主莖切片PPO活性皆趨近於0 U·μg-1protein，至處理後72小時才可測得PPO活性。LsPALs基因表現部分，醋酸與正丁醇皆能抑制LsPALs基因表現，其中又以醋酸的抑制效果為佳，而CysH則可延後LsPALs基因表現至處理後48小時上升。

上述結果顯示由於醋酸能同時抑制PAL蛋白質之生合成與其活性，故擁有最佳之褐變抑制效果。CysH處理亦可延後PAL之基因表現與活性上升，並完全抑制PPO之酵素活性，故亦有良好之褐變抑制效果。而正丁醇僅能抑制LsPALs基因表現，於長時間儲藏條件下PAL活性仍有上升之現象，使得正丁醇處理之褐變抑制效果較差。

醋酸雖然具有最佳之褐變抑制效果，然而部分樣本在醋酸處理後12天出現微生物孳生之狀況。因此，將其結合具有抑菌效果之酒精處理進行測試。結果顯示先施用酒精再立即進行醋酸處理與兩處理合併使用，此兩種方式皆能有效抑制褐變，抑制褐變效果皆與僅施用醋酸相似，而單獨施用酒精處理抑制褐變效果較差。施用合併處理進行生菌之觀察，處理後14天仍未出現微生物生長，且於處理後14天生菌數皆低於單獨施用醋酸處理。因此判斷醋酸及酒精合併處理可以減緩微生物生長現象，故可利用此處理來解決減緩結球萵苣底部褐化的問題，延長其櫥架壽命。

Iceberg lettuce (Lactuca sativa L. var. capitata) is an annual plant in Compositae family. Nowadays iceberg lettuce becomes an important crop for export in Taiwan. However, the cut surface of iceberg lettuce would turn red brown after harvest. It may cause the quality loss and shorten the shelf life. The browning resulted from oxidation of polyphenol contents which was catalyzed by polyphenol oxidase (PPO). Besides, it was known that wounding would induce the phenylalanine ammonia lyase (PAL) activity of lettuce stem disks and cause the phenolics contents increasing. Therefore, the induction of PAL activity was considered to be the key point of lettuce browning. Therefore, the objective of this research was to elucidate the relationship between PAL homologs and lettuce butt discoloration in transcript level.

With the latest genome database of lettuce, Lettuce Genome Resource (LGR), we screened out six sequences with PAL annotation. Then using specific primers to do full-length PCR, we successfully identified Lsa027790.1 and Lsa019010.1 with gDNA template; amplified Lsa005270.1, Lsa027790.1, and Lsa019025.1 with cDNA template. Besides, according to the similarity to published LsPAL1 to give them the names from LsPAL1 to LsPAL5 and LsPAL5 has two isoforms. Then, use semi quantitative PCR to examine the expression patterns and the results suggested that LsPAL1 to LsPAL4 were induced in 2 hrs after wounding and reached the peak in 8 hrs after wounding. LsPAL5 showed no expression before and after wounding yet. Consequently, we could know that LsPALs might play an important role in butt discoloration except for LsPAL5.

There are several browning inhibitors which are effective on inhbiting butt discoloration shown in previous study. Then we applied these browning inhibitors on the cut surface to characterize the role each member of LsPAL gene family played. Besides, we also revealed the inhibition mechanism of each inhibitor and what kind of treatment was proper on butt discoloration. After soaked in 3% acetic acid for 5 seconds after wounding, stem disks maintained original color to 10 days. Three percent of 1-butanol treatment delayed the lettuce browning happened but the stem disks still turned brown in 10 days after treatment. Three percent of L-cysteine hydrochloride (CysH) treatment also inhibited browning but the stem disks would turn yellow in 10 days after treatment. In addition, acetic acid effectively inhibited PAL activity in 72 hours after treatment. Similarly, 1-butanol and CysH treatments delayed the inducition of PAL activity. Besides, acetic acid and 1-butanol treatment increased the PPO activity; whereas, CysH totally inhibited the PPO activity in 48 hours after treatment. In transcript level, both acetic acid and 1-butanol treatment repressed the LsPALs gene expression and CysH treatment delayed the induction of LsPALs to 48 hours after treatments.

To sum up, due to the repression on PAL synthesis and activity, acetic acid treatment was thought to be the most effective approach to inhibit enzymatic browning on lettuce stem disks among these three antibrowning agents. CysH treatment not only delayed the induction of PAL gene expression and activity but also inhibited the PPO activity, and thus had a good atibrowning effect. Treating 1-butanol only inhibited LsPALs gene expression while PAL activity in lettuce stem disks still increased. It resulted in 1-butanol treatment having less antibrowning effect on lettuce stem disks after storage for long time.

Although acetic acid treatment could totally inhibit the browning, it would cause severe microbial growth on lettuce stem disks in 12 days after acetic acid treatment. Therefore, we combined ethanol treatment and acetic treatment to test whether this combination treatment could repress browning and reduce microbial growth simultaneously. The results showed that both of applying ehanol first then using acetic acid treatment and combining this two chemicals at the same time could effectively retard browning. The antibrowning effect of combination treatment is better than ethanol treatment. Besides, there is no visible microbial growth for 2 weeks after applying combination treatment. Additionally, the microbial counts on combination treatment treated lettuce stems were still lower than those of the acetic treatment. These results showed that combination of acetic acid and ethanol treatment had high potential to be applied to solve the butt discoloration problem and extend the shelf life.

口試委員會審定書 #
誌謝 i
中文摘要 ii
ABSTRACT iv
目錄 vi
圖目錄 ix
表目錄 x
前言 1
第一章 前人研究 2
第一節 萵苣簡介 2
第二節 臺灣結球萵苣產業概況 3
第三節 結球萵苣切面褐化發生機制 5
(一) 酚類化合物之生合成 6
(二) 苯丙胺酸氨基裂解酶 6
(三) 多酚氧化酶 7
第四節 延緩萵苣褐變之處理技術 8
(一) 物理性採後處理技術 9
(二) 化學性採後處理技術 10
(三) 其他處理方式 11
第二章 材料與方法 12
第一節 植物材料與處理 12
(一) 植物材料與主莖切片方式 12
(二) 藥劑處理方式 12
(三) 萵苣主莖切片外觀調查 12
第二節 結球萵苣LsPAL基因家族序列取得與驗證 13
(一) 去氧核醣核酸萃取 (DNA isolation) 13
(二) 核糖核酸萃取 (RNA extraction) 14
(三) 去氧核醣核酸酶(DNase)處理 14
(四) 反轉錄(reverse transcription) 14
(五) 基因全長聚合酶連鎖反應(polymerase chain reaction, PCR) 15
(六) DNA膠體電泳(gel electrophoresis)分析 15
第三節 親緣關係樹(phylogenetic tree)建立 16
(一) 胺基酸多重序列比對(multiple sequence alignment) 16
(二) 親緣關係樹建立 16
第四節 結球萵苣LsPAL基因家族基因表現 16
(一) 半定量PCR 16
(二) 即時定量聚合酶連鎖反應(Real-time quantitative polymerase chain reaction, qPCR) 16
第五節 PAL與PPO活性測定 17
(一) 藥品製備 17
(二) PAL與PPO酵素萃取 18
(三) PAL與PPO活性測定 18
(四) 酵素粗萃取液總蛋白質含量定量 18
第六節 結球萵苣主莖切片總生菌數測定 19
第七節 數據作圖與統計分析 19
第三章 結果 20
第一節 創傷與結球萵苣主莖切面酵素型褐變之關聯 20
第二節 結球萵苣LsPAL基因家族序列取得與驗證 20
第三節 LsPALs 基因序列分析 21
第四節 LsPALs於結球萵苣主莖切面褐變下之基因表現 21
第五節 褐變抑制處理對結球萵苣主莖切面褐變之影響 22
第六節 褐變抑制處理對結球萵苣主莖切片PAL與PPO活性之影響 23
第七節 褐變抑制處理對LsPALs基因表現之影響 24
第八節 醋酸與酒精複合處理對結球萵苣主莖切面褐變之影響 25
第四章 討論 27
第一節 PAL為結球萵苣主莖切面褐變之關鍵控制點 27
第二節 LsPAL基因家族與結球萵苣主莖切面褐化具直接關聯性 27
第三節 抑制褐變處理對LsPAL基因家族與結球萵苣主莖切面褐變之影響 29
(一) 醋酸處理與主莖切面褐變之關係 29
(二) 正丁醇處理與主莖切面褐變之關係 30
(三) 半胱胺酸鹽酸鹽處理與主莖切面褐變之關係 31
第四節 酒精與醋酸複合處理與結球萵苣主莖切面生菌數之關係 32
第五章 結論 34
結果圖表 35
參考文獻 61

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