Responses of Growth and Antioxidant Defense System of Corn to the Acid Stress

本試驗主要在於探討人工模擬酸性逆境對玉米種子發芽及三葉期幼苗生長兩個生育階段的影響。 將玉米種子以不同pH值分別為2.0、2.5、3.0、4.0、5.0和5.5之酸性溶液處理，進行發芽試驗，結果顯示pH 2.5和2.0酸性溶液明顯抑制種子發芽、胚根和芽鞘的生長。此外，pH 2.5的酸性逆境明顯抑制發芽種子α-amylase活性、澱粉分解、蔗糖和葡萄糖的合成；種子中並累積較多的H2O2和 MDA。由上述結果推測，在酸性逆境下發芽種子發生過氧化作用，造成氧化傷害，結果抑制α-amylase活性，導致澱粉分解受阻，進而阻礙玉米種子正常的發芽。 玉米三葉期幼苗分別以pH 2.0、2.5、3.0、4.0、5.0和5.5之酸性溶液處理，結果顯示pH 2.5和2.0酸性溶液抑制株高的生長、根部及地上部的乾物重和相對生長速率。在pH 2.5酸性逆境下，幼苗葉片葉綠素含量明顯減少，但葉綠素a/b比值並無明顯改變；pH 2.5酸性逆境下葉片葉綠素螢光釋放量較高，顯示光合作用受抑制；此外，幼苗葉片細胞發生大量離子滲漏，顯示葉片細胞之原生質膜受損。在pH 2.5酸性逆境下葉片和根部的H2O2和MDA含量均提高，顯示植株發生過氧化作用及氧化傷害；另一方面，葉片和根部抗氧化防禦系統呈現Asc含量下降、APx活性降低、GSH含量增加、GSH/GSSG比值提高和GR活性增加的反應趨勢。由上述試驗結果推測，酸性逆境可造成植物發生過氧化作用及氧化傷害，進而引起葉片光化學活性降低，葉綠素含量減少，細胞原生質膜及胞內膜系受損，導致葉片提早老化，乾物質合成減少，最後影響幼苗的生長。The purpose of this experiment is to explore the effect of stimulated acid stress on germination and V3-seedling growth of maize. Maize seeds were treated with acid solutions of different pH values, i.e. 2.0, 2.5, 3.0, 4.0, 5.0, and 5.5, respectively. The results showed that the acid solutions of pH 2.5 and 2.0 had a significant decrease in germination rate, radicle and coleoptile growth. In addition, the acid stress of pH 2.5 decreased significantly in α-amylase activity, starch decomposition, glucose and sucrose synthesis in germinating seeds; also accumulated more H2O2 and MDA. It postulated that under acid stress the germinating seeds suffered from oxidative damage, and then resulted in the decrease of α-amylase activity and starch decomposition. Consequently, the germination was depressed. V3-seedlings of maize were treated with acid solutions of different pH values, i.e. 2.0, 2.5, 3.0, 4.0, 5.0, and 5.5, respectively. The results showed that acid solutions of pH 2.5 and 2.0 had a significant depression to the plant height, dry weight and RGR of roots as well as above-ground parts. Under the acid stress of pH 2.5, the chlorophyll content in seedling leaves decreased, however, the ratio of chlorophyll a/b didn't show a significant change. Under acid stress of pH 2.5, the leaf maintaining a higher release of the chlorophyll fluorescence indicated that the photosynthesis of seedling was inhibited. The acid stress of pH 2.5 also induced a greater ion leakage. A serious peroxidation and oxidative damage had been obserbed in the maize seedling as subjected to acid stress of pH 2.5. Antioxidant defense system had also been tested. Asc content and APx activity decreased, whereas GSH content, GSH/GSSG ratio, and GR activity were increased in both leaves and roots of seedling. Based on the aboved results, it's concluded that peroxidation and oxidative damage can be occurred under the acid stress, and induce photochemical activity depression, chlorophyll loss, membrane permeability increase, premature senescence of leaves, and dry matter synthesis inhibition, subsequently the growth of seedling was depressed.中文摘要…………………………………………………………… i 英文摘要…………………………………………………………… ii 目錄………………………………………………………………… iii 表目錄……………………………………………………………… iv 圖目錄……………………………………………………………… v 縮寫字……………………………………………………………… vii 緒言………………………………………………………………… 1 前人研究…………………………………………………………… 4 材料與方法………………………………………………………… 20 結果………………………………………………………………… 32 第一部分：酸性逆境對玉米種子發芽之影響…………………… 32 試驗一：不同pH值酸性溶液對玉米種子發芽之影響…………… 32 試驗二：酸性逆境對玉米發芽種子澱粉分解之影響…………… 40 試驗三：酸性逆境造成玉米發芽種子的氧化傷害……………… 44 第二部分：酸性逆境對玉米三葉期幼苗生長之影響…………… 49 試驗一：不同pH值酸性溶液對玉米幼苗根部及地上部生長之影響…………………………………………………………………… 49 試驗二：酸性逆境對玉米幼苗光合作用和葉片老化之影響…… 56 試驗三：酸性逆境對幼苗葉片氧化傷害及抗氧化反應………… 62 試驗四：酸性逆境對幼苗根部氧化傷害及抗氧化反應………… 73 討論………………………………………………………………… 88 參考文獻…………………………………………………………… 9

Studies on Transferring Transglutaminase Gene into Rice Chloroplast

本論文之目的為：(1) 增進水稻成熟種子癒傷組織誘導再生芽體、(2) 建立篩選抗生素及D-胺基酸對水稻種子發芽和癒傷組織芽體再生之抑制濃度、(3) 應用葉綠體轉殖水稻表現轉榖氨醯胺酵素基因、(4) 建立剔除篩選基因水稻表現轉榖氨醯胺酵素。 本研究採用水稻成熟種子所誘導的癒傷組織作為水稻葉綠體基因轉殖之培殖體，並探討維生素、有機氮源、植物生長調節劑等對水稻癒傷組織誘導再生芽體之影響。維生素試驗顯示MS維生素處理有較高'每盤芽體數'主要經由增加'每盤形成芽體之癒傷組織數'。有機氮源試驗顯示以不添加有機氮源處理組可誘導產生最多再生芽體，其次依序為casamino acid、tryptone、peptone。''台農67號'' (''TN 67'')及''台稉9號'' (''TK9'')二品種間對於自癒傷組織誘導再生芽體並無顯著差異，且均有良好再生芽體誘導效率。 TDZ、BA和IAA三種植物生長調節劑影響水稻癒傷組織誘導再生芽體之試驗結果顯示，在20個組合中有9個組合每盤芽體數大於50個，其中以A12組有最高的再生芽體誘導效率，其植物生長調節劑之組合為0.5 mg/L TDZ、2.0 mg/L BA、0.1 mg/L IAA。多變量分析(multivariate analysis of variance, MANOVA)顯示，TDZ、BA、IAA對水稻癒傷組織誘導再生芽體呈顯著影響(p < 0.01)，且TDZ和IAA間、BA和IAA間二項亦顯著呈現具有交感效應(p < 0.01)。BA和IAA之交感效應顯示，2 ppm BA以搭配0.1 ppm IAA、3 ppm BA以搭配0.2 ppm IAA有較多的'每盤芽體數'，推算BA和IAA之比例分別為20: 1、15: 1時可誘導較多芽體形成，且顯示在較低濃度時，需較高的BA/IAA比例。 本研究測試不同濃度spectinomycin、streptomycin、D-alanine對水稻種子發芽、再生芽體誘導之抑制效果，以作為葉綠體基因轉殖採用篩選濃度之參考依據。篩選試劑對於水稻種子發芽之影響，以200 ppm streptomycin對水稻種子發芽明顯呈現抑制作用，可作為篩選濃度之參考。500 ppm D-alanine (5.6 mM)可以有效抑制水稻種子發芽，此濃度可應用於葉綠體基因轉殖之篩選。對於水稻癒傷組織誘導再生芽體之影響，當spectinomycin濃度增加至700 ppm時，每個培養皿仍可再生22個芽體，抑制效果不佳。當streptomycin濃度200 ppm時，明顯抑制再生芽體，所誘導的芽體白化情形明顯，黃化或白化的癒傷組織和芽體可作為篩選的抑制指標。200 ppm (2.2 mM) D-alanine處理抑制芽體再生且造成癒傷組織明顯褐化，可為篩選之依據。 轉穀氨醯胺酵素(transglutaminase, TGase)可催化蛋白分子內或蛋白間形成類胜肽鍵結(isopeptide bond)，主要應用在食品、醫藥和紡織工業。本研究經由葉綠體基因轉殖利用水稻葉綠體作為分子農場表現轉穀氨醯胺酵素(transglutaminase, TGase)。pMT92-EPA和pMT92-ETA二個轉殖載體各自經基因槍轟撃2,000個水稻癒傷組織，經過streptomycin篩選，分別獲得14和20株芽體再生之水稻植株，經PCR和RT-PCR分析，其中各有1和3株具有轉殖基因及其表現，包括tga、eGFP、aadA，水稻植株編號分別為EPA-5和ETA-4、12、18。利用數位影像系統Kodak image station 4000MM進行eGFP螢光分析，EPA-5和ETA-4、12、18水稻葉片可偵測到eGFP螢光表現。葉綠體基因組遺傳為母系遺傳，將採收後種子(T1)進行發芽篩選試驗。經PCR分析，顯示轉殖基因tga、eGFP、aadA可傳遞至ETA-18之T1子代ETA-18_2, 6, 7。 本研究利用葉綠體基因轉殖之標記基因剔除技術，建立表現轉穀氨醯胺酵素(transglutaminase, TGase)之無篩選基因葉綠體基因轉殖水稻，並以環狀和直線狀DNA二種轉殖載體進行基因槍轟擊。以直線型載體pMT92GP-sA進行轉殖，獲得7株再生水稻植株。經PCR和RT-PCR分析，僅檢測到1株GP-sA-lin-3具有轉殖基因tga、gus、aadA，且偵測到三個基因表現，顯示並未將篩選基因aadA剔除。 環狀pMT92GP-sDA轉殖載體部分，分別以streptomycin或D-alanine篩選，各獲得23、17株再生水稻植株。PCR分析結果顯示，利用streptomycin篩選有二株(GP-sDA-str-7、21)具有轉殖基因tga、gus、aadA、daao。RT-PCR分析亦偵測轉殖基因表現，顯示二株水稻植株均並未剔除篩選基因aadA、daao。利用D-alanine篩選所得之水稻植株，PCR與RT-PCR分析顯示有二株(GP-sDA-ala-8、17)具有轉殖基因tga、gus及其表現，其中GP-sDA-ala-8仍具篩選基因aadA、daao及其表現，而GP-sDA-ala-17則未檢測出篩選基因aadA、daao。GUS組織化學之活性染色分析顯示4株水稻均呈現GUS活性染色反應。從所獲得的水稻植株與具有轉殖基因植株數，streptomycin和D-alanine篩選準確率分別為8.7和11.8%，其中經D-alanine篩選之GP-sDA-ala-17顯示剔除篩選基因。The purpose of this thesis includes: (1) Optimization for shoot regeneration from rice mature seed-derived callus, (2) Establishment of inhibitory concentration of selection agents on seeds germination and callus regeneration of rice, (3) Application of transplastomic rice to express transglutaminase gene, (4) Toward establishing an efficient selectable marker elimination system for stable expression of transglutaminase in transplastomic rice. In this thesis, the callus derived from matured seed of rice were used as the explants of biolistic chloroplast transformation. The effects of vitamins, organic nitrogen sources and plant growth regulators on the shoots regeneration from rice callus were investigated. In the vitamins comparison, the results indicated that the contribution of MS vitamins to the 'shoot number per plate' was mainly influenced through the 'percentage of shooting callus'. Regarding the organic nitrogen sources, the maximum 'shoot number per plate' could be induced without organic nitrogen source, followed by casamino acid, tryptone and peptone. About the rice cultivars Tainung 67 and Taiken 9, there was no significant difference between the two cultivars on the shoot regeneration, and both have good performance. Different combinations of plant growth regulators, TDZ, BA and IAA, were used to induce shoots from rice callus. The results showed that there were nine combinations can induce more than 50 shoots per plate in 20 combinations. Among them, A12 has the highest induction efficiency of regenerated shoots, the combination of its plant growth regulators includes 0.5 mg/L TDZ, 2.0 mg/L BA and 0.1 mg/L IAA. MANOVA showed that TDZ, BA and IAA had significant effects on the induction of shoots from rice callus (p < 0.01). Interaction effect between TDZ and IAA, BA and IAA also showed significant (p < 0.01). The interaction effect analysis of BA and IAA revealed that 2 ppm BA with 0.1 ppm IAA, 3 ppm BA with 0.2 ppm IAA can have more 'shoot number per plate'. The ratio of BA and IAA is estimated to be 20: 1 and 15: 1, and shows a higher BA/IAA ratio at lower concentrations. For the screening of chloroplast transformants, the inhibitory effects of different concentrations of spectinomycin, streptomycin and D-alanine on seed germination, regenerated shoots induction were tested. On the effects of inhibition rice seed germination, 200 ppm streptomycin is sufficient to inhibit the germination of rice seeds, which could be used as a reference for selection concentration. D-alanine can effectively inhibit rice germination at 500 ppm (5.6 mM), this concentration can be applied to the selection of chloroplast transformants. The result of spectinomycin on the inhibition of shoot induction from rcie callus indicate that, when the concentration was increased to 700 ppm, there were still 22 shoots per plate, the inhibition effect was poor. In addition, there were significantly inhibition on regenerated shoot when streptomycin was 200 ppm, the formation of albino shoot was more obvious, yellow or whitening calli and shoots can be used as an indicator of selection. While shoot induction from callus subjected to D-alanine, 200 ppm (2.2 mM) inhibited the shoot regeneration and resulted in the browning of callus, can be used as working concentration. Transglutaminase (TGase) can catalyze the formation of an isopeptide bond, has been applied in food, pharmaceuticals and textiles industry. In this study, the transplastomic rice was used as a molecular farming to express TGase. The transgenic vectors pMT92-EPA and pMT92-ETA were each used to bombard 2,000 rice calli by gene gun. After selection with streptomycin, 14 and 20 regenerated rice plants were obtained respectively. Each of them has 1 and 3 lines, respectively EPA-5 and ETA-4, 12, 18, can detect the transgenic genes and transgene expression, including tga, eGFP and aadA by PCR and RT-PCR. The eGFP fluorescence can be observed also in the leaves of EPA-5 and ETA-4, 12, 18 by using Kodak image station 4000MM. The genetics of the chloroplast genome are maternal inheritance. The harvested T1 seeds were subjected to germination selection. According to the PCR analysis, transgenic genes of tga, eGFP and aadA were stably transmitted to ETA-18 progeny, including ETA-18_2, 6, and 7 plants. In this study, the marker gene elimination technique of chloroplast gene transfer was used to establish a marker free transplastomic plant for TGase gene expressing. The transgenic vectors were divided into two forms of circular and linear DNA. The linear transgenic vectors pMT92GP-sA was used for bombardment, and obtained 7 lines of regenerated rice. PCR and RT-PCR analysis showed that one plant GP-sA-lin-3 was detected only in the regenerated rice of pMT92GP-sA with the transgenic gene tga, gus and aadA, and their expression. Indicating that the selection gene aadA was not removed. The transgenic plants of circular pMT92GP-sDA were selected with streptomycin or D-alanine respectively. Each of which produces 23 and 17 regenerated rice plants. PCR analysis showed that two lines of GP-sDA-str-7 and 21 obtained by streptomycin screening contained the transgenic gene tga, gus, aadA and daao. RT-PCR analysis also detected the expression of all transgenic genes, indicating that two rice plants did not eliminate the selectable marker gene aadA and daao. The PCR and RT-PCR analysis of rice plants obtained by D-alanine selection showed that two plants GP-sDA-ala-8 and 17 had the transgenic gene tga and gus, and their expression. Among them, GP-sDA-ala-8 still had selection genes aadA, daao and its expression, but not detected in GP-sDA-ala-17. GUS staining analysis showed that the four strains of rice showed GUS activity. The correct rate of streptomycin and D-alanine selection was 8.7 and 11.8%, respectively, from the number of regenerated rice plants and the number of plants with transgenic genes. The GP-sDA-ala-17 selected by D-alanine showed that the selection marker gene had been eliminated.中文摘要……………………………………………………………i 英文摘要…………………………………………………………iii こ 緒言……………………………………………………………………… 1 第一章、前人研究………………………………………… 5 第二章、水稻成熟種子癒傷組織誘導再生芽體之研究 中文摘要…………………………………………………………… 22 英文摘要…………………………………………………………… 23 前言……………………………………………………………………… 24 材料與方法……………………………………………………… 25 結果……………………………………………………………………… 27 討論……………………………………………………………………… 64 第三章、篩選試劑抑制水稻種子發芽和癒傷組織芽體再生之研究 中文摘要…………………………………………………………… 70 英文摘要…………………………………………………………… 71 前言……………………………………………………………………… 72 材料與方法……………………………………………………… 73 結果……………………………………………………………………… 75 討論……………………………………………………………………… 89 第四章、應用葉綠體轉殖水稻表現轉榖氨醯胺酵素基因之研究 中文摘要…………………………………………………………… 94 英文摘要…………………………………………………………… 95 前言……………………………………………………………………… 96 材料與方法……………………………………………………… 98 結果……………………………………………………………………… 105 討論……………………………………………………………………… 130 第五章、剔除篩選標誌基因系統應用於葉綠體轉殖水稻表現轉榖氨醯胺酵素基因之研究 中文摘要…………………………………………………………… 134 英文摘要…………………………………………………………… 135 前言……………………………………………………………………… 136 材料與方法……………………………………………………… 138 結果……………………………………………………………………… 145 討論……………………………………………………………………… 173 結論……………………………………………………………………… 176 參考文獻…………………………………………………………… 17

轉殖溶菌酶(Lysozyme)及穿孔素(Holin)基因增加水稻抗病之研究

本研究將分離自 Xanthomonas fragariae (草莓角斑病菌)菌株的類似噬菌體(phage XF)的溶菌酶 (lys)及穿孔素 (hol)基因，並構築到水稻葉綠體基因轉殖之二種載體(pMT92-GHA 及 pMT92F-GL-sA)，利用基因槍轟擊法，混合轉殖到'台農 67 號'水稻之癒傷組織的葉綠體。培殖體經 100 ppm streptomycin 持續篩選三個月，可獲得再生植株。再生的水稻葉片以 PCR 及 RT-PCR 分析之結果顯示，lys 基因、hol 基因及 gus 報導基因，已存在於轉殖水稻植株的葉綠體基因組中，並表現其 mRNA。轉殖水稻葉片接種水稻白葉枯病菌後，明顯的具抗病的特性。本研究初步結果顯示，利用水稻葉綠體基因轉殖系統，共同轉殖 lys 基因及 hol 基因，培育出抗病水稻是可行的。In this study, two chloroplast transformation vectors, designated as pMT92-GHA and pMT92F-GL-sA, which contained phage lysozyme gene (lys) and holin gene (hol) as target genes, respectively, were transferred into rice chloroplast via biolistic bombardment. The regenerated plants were primarily selected by 100 ppm streptomycin and further confirmed by PCR and RT-PCR. The results of PCR and RT-PCR analysis indicated that transformed lys, hol, and gus genes were integrated into the plastid genome of transplastomic plants, and its mRNAs were expressed. Resistance to bacterial leaf blight of rice was found in the lys and hol gene transplastomic plants after inoculation with Xanthomonas campestris pv. oryzae