Molecular Breeding of Sesame Nutritive Rice

動物所需之必需胺基酸並不能自行合成，因此必須藉由採食中獲得。而就胺基酸組成而言，植物性蛋白質比動物性蛋白質在甲硫胺酸（methionine）與離胺酸（lysine）的含量明顯偏低。因此，以植物為主要食物來源的動物（含人類），甲硫胺酸與離胺酸較易缺乏。動物體內雖可以把甲硫胺酸轉變成胱胺酸，但此路徑卻不可逆，即甲硫胺酸可提供完整的含硫必需胺基酸，但胱胺酸卻不能。因此，動物對含硫胺基酸的吸收必須要獲得甲硫胺酸最低限度的量。本研究主要目的為利用農桿菌基因轉殖技術，將芝麻種子中富含甲硫胺酸之2S清蛋白的對應基因轉殖於水稻基因體中，預期能育成具高營養及附加價值的新品種水稻。於表現載體構築中，選擇以水稻種子中之穀蛋白（glutelin）及油體膜蛋白（oleosin）特異表現之啟動子攜帶芝麻2S清蛋白基因，在種子成熟時，能於米粒中及/或米糠（含胚芽）中表達此外來基因，此結果除了可增加稻米之營養品質外，稻米碾白後之副產物（米糠），其高甲硫胺酸含量即可應用在飼料工業上。以穀蛋白為啟動子之結果，已將芝麻2S清蛋白表現於米粒，此品種命名為「芝麻營養米」。另一方面，以油體膜蛋白為啟動子之轉殖水稻，芝麻2S清蛋白僅特異表現於米糠，此品種稱為「二代芝麻營養米」。 另外，也利用單一表現載體，探討同時將兩個重組蛋白於植物細胞中表現。此載體之構築，以水稻種子中之穀蛋白為啟動子，攜帶包含芝麻油體膜蛋白及其C端再接上2S清蛋白之全長對應基因。結果顯示，此重組蛋白質可於成熟水稻種子之內質網中發現，其分子量約為32 kDa (油體膜蛋白為 17 kDa 與 2S清蛋白為15 kDa)。在水稻種子成熟後，芝麻油體膜蛋白及2S清蛋白則分別座落於水稻之油體 (oil bodies) 及蛋白體 (protein bodies)。Animals are unable to synthesize essential amino acid, and have to acquire this essential amino acid from their food. Since animals are capable of converting methionine to cysteine, but not the reverse, methionine is the only essential S-amino acid in terms of food nutrition. Dietary deficiency of methionine results in an imbalanced uptake of amino acids and may retard the growth and development of human and animals. Based on the deduced amino acid sequence of its corresponding gene, 2S albumin, the major soluble protein in sesame seed has been demonstrated to be a sulfur-rich protein, which apparently accounts for the high quality of nutritive content in sesame. In light of the limited nutritive value of rice caused by its low S-amino acid content, I have achieved the molecular breeding of rice by introducing the edible sesame sulfur-rich protein via transgenic technique. The transgenic rice plants specifically accumulate sesame 2S protein in their grain or bran by the control of glutelin or oleosin promoters. Now, I achieved these transgenic lines, and named individually by ‘Sesame Nutritive Rice' and ‘Second Generation Sesame Nutritive Rice'. Moreover, a new technique introduced two recombinant polypeptides separately assembled into rice oil bodies and protein bodies in one expression construct. A recombinant polypeptide containing the precursor protein of a sesame storage protein, 2S albumin, fused to the C-terminus of a sesame oleosin was expressed in transgenic rice seeds under the control of a rice glutelin promoter. The recombinant polypeptide of 32 kDa, equivalent to the resultant molecular mass of sesame oleosin (15 kDa) and prepro-2S albumin (17 kDa), was detected in the ER fraction of maturing transgenic rice seeds. In mature transgenic seeds, sesame oleosin was found in purified oil bodies while mature sesame 2S albumin was detected in grain extract. Immunogold labeling revealed that the sesame oleosin and 2S albumin were separately localized in oil bodies and protein bodies of transgenic rice seeds.Chinese abstract…………………………………………………………………1 English abstract………………………………………………………………….3 List of Contents…………………………………………………………………..5 List of Figures……………………………………………………………............ 6 List of Tables………………………………………………………………………8 Abbreviations……………………………………..………………………………9 Research background…………………………………………………………10 Chapter 1. Accumulation of a sesame 2S albumin enhances methionine and cysteine levels of transgenic rice seeds………………………………………………………………...18 Chapter 2. Specific expression of a sesame storage protein in transgenic rice bran…………………………………………….42 Chapter 3. Sesame oleosin and prepro-2S albumin expressed as a fusion polypeptide in transgenic rice were split, processed and separately assembled into oil bodies and protein bodies……………………………………………………………….62 Conclusion……………………………………………………………………….8

Study of protease digestion on various feed proteins

本試驗之主要目的為：一、利用化學分析法評估不同蛋白對飼料蛋白質之作用；二、以動物試驗探討外源蛋白對飼料蛋白質於肉雞之消化效果。試驗一，以對酪蛋白具有相同活性（1.25unit/ml）之Streptomyces caespitosus蛋白及鳳梨酵素，作用於酪蛋白、大豆粕、魚粉、濃縮大豆蛋白（soya protein concentrate）及玉米麩質（corn gluten meal）等飼料蛋白質不同時間後，測定酪胺酸釋出量、蛋白作用後殘留物氮含量、可溶性蛋白質含量及蛋白分佈圖譜。結果顯示，鳳梨酵素作用飼料蛋白質之效果顯著較Streptomyces caespitosus蛋白為佳。評估蛋白對飼料蛋白質之作用程度，如由標準測定蛋白活性高低之酪胺酸釋出量結果與其他測定值所得之結果，呈不一致之趨勢。因以作用產物之酪胺酸釋出量多寡，並不能完全代表酵素作用後之產物型態，故以酪蛋白為受質及酪胺酸釋出量所測得蛋白之活性方法，直接應用在評估外源蛋白作用飼料蛋白質之活性並不恰當。 試驗二，以生長期（21日齡）及肥育期（42日齡）之Arbor Acres種肉公雞120隻與60隻，個別飼養於代謝籠，並逢機分配於10個處理組。處理為含酪蛋白、大豆粕、魚粉、濃縮大豆蛋白及玉米麩質等蛋白質來源配製半純化飼糧及有無添加鳳梨酵素之5×2複因子設計。進行指示劑法之表面迴腸蛋白質消化率（Apparent ileal protein digestibility, AIPD）測定及食糜性狀分析。由生長期及肥育期之結果顯示，鳳梨酵素之添加對AIPD（%）並無改善之效果，且於消化道食糜總蛋白及可溶性蛋白質之分子量分佈圖譜顯示，鳳梨酵素之有無添加或不同生長期間，所獲得之圖譜均相似，顯示添加外源鳳梨酵素並未能改善不同飼料蛋白質於肉雞消化道之消化效果，可能因鳳梨酵素於肉雞之肌胃內即失去活性所致。不同飼料蛋白質之AIPD（%）以酪蛋白組最佳，其次為魚粉、濃縮大豆蛋白、大豆粕組，而玉米麩質組最差（P10kDa之蛋白質逐漸被水解，而分子量<10kDa者逐漸增加，因酪蛋白組之可溶性蛋白質由大分子量改變之比例最為明顯，顯示其蛋白質被水解程度最快，玉米麩質最慢，因大豆粕及濃縮大豆蛋白組成類似，故於雞隻消化道食糜可溶性蛋白質分子量之分佈圖譜一致。由以上結果得知，鳳梨酵素添加於含不同飼料蛋白質之飼糧並不能改善肉雞對飼料蛋白質之利用，而由不同飼料蛋白質於肉雞消化道食糜可溶蛋白質分子量改變速率與測得之AIPD（%）具一致之結果，顯示飼料蛋白質於腸道之水解速率與其消化率有關。 利用化學分析法評估外源蛋白對飼料蛋白質之作用結果，以作用產物之酪胺酸釋出量多寡，並不能完全代表酵素作用後之產物型態，需配合總蛋白質水解情形（可溶性蛋白質、殘留物含氮量及蛋白分佈）方能判定。而外源蛋白之實際生物可利用性，則需進行動物試驗，方能更準確獲知其應用價值。The aims of this study were to evaluate the activity of protease digestion to various feed proteins sources in “Chemical assay&quot;, and the effect of protease supplement in diet containing different protein sources on digestion of broilers. In trial 1, feed proteins include casein, soybean meal, fish meal, soya protein concentrate and corn gluten meal were treated with same unit (1.25U/ml) of enzyme from Streptomyces caespitosus and Bromelain. From the concentration of the tyrosine released, nitrogen of residue, soluble protein and polypeptide pattern by SDS-PAGE after different incubation times, it showed that the activity of Bromelain were better significantly than Streptomyces caespitosus protease, and the hydrolyzed activity to different feed protein had the same trend in all measurement except tyrosine releasing level. The amount of tyrosine released couldn't represent residue pattern of protein after enzyme digestion. Therefore, using casein as substrate and tyrosine releasing method couldn't directly to evaluate protease activity for other feed proteins. In trial 2, 120 twenty-one days-old and 60 forty-two days-old broilers (Arbor Acres) were individually allocated in metabolism cages and randomly divided into 10 groups with a 5×2 factorial design arrangement for digestibility and digesta measurements. Treatment groups included casein, soybean meal, fish meal, soya protein concentrate and corn gluten meal as diet protein source to adjust semi-purified diet and with or without Bromelain supplement. The result showed that there were no improvements in AIPD (%). The total protein peptides and soluble protein pattern of digesta were also not significant different in Bromelain supplement in grower or finisher broiler diet. Compared with different feed proteins, casein group had the best AIPD (%), followed with the fish meal, soya protein concentrate and soybean meal group, corn gluten meal group was significantly lower than others (P<0.05), the value were 92.1, 83.8, 85.5, 84.1 and 76.3% respectively. The digesta of gizzard had the highest pH value in casein group, which was because casein was hydrolyzed by pepsin easier and had a higher buffer capacity than others in gizzard. The soluble protein in digesta from different treatment group showed different pattern. However, the peptides with molecular weight above 10kDa were hydrolyzed and below 10kDa were increased gradually in all of groups except in the corn gluten meal group. The soluble protein pattern in digesta of casein group changed significantly, which represented that casein hydrolyzed faster and easier, in opposite to corn gluten meal which was hydrolyzed slowest. The digesta had the same distribution of protein molecular weight in soybean meal and soya protein concentrate groups because they contained the same protein constituent. Here we concluded that supplement Bromelain in semi-purified with various protein sources couldn''t improve protein utilization in broilers. The distribution pattern of soluble protein in gastrointestinal digesta and the value of AIPD had the same trend; it showed the hydrolyzed rate of feed protein sources in gastrointestine tract could affect the protein digestibility in broilers. Using &quot;Chemical assay&quot; to evaluate the protease activities in different feed protein sources, it couldn''t only consider enzymes digestion pattern by tyrosine released. It must to consider the hydrolyzed product of total protein (the level of soluble protein, residue and polypeptide pattern), and exogenous enzyme utilization must use &quot;Animal trial&quot; to achieve apply merit more accuracy.目 錄 頁次 壹、中文摘要 1 貳、前言 3 參、文獻檢討 5 一、蛋白質在體內消化過程 5 二、蛋白質在體內吸收與代謝 5 三、影響飼料蛋白質利用之因子 5 （一）飼料蛋白質製備方式 6 （二）加工處理及方式 6 （三）飼糧中纖維組成分之影響 6 四、以化學分析法（試管內）評估蛋白對蛋白質之作用 7 （一）水解產物氮含量變化 7 （二）水解產物之蛋白圖譜變化 8 五、以動物試驗評估不同蛋白質被消化之情形 8 （一）食糜特性之變化 8 （二）消化率之測定 9 六、蛋白的來源 10 七、蛋白之測定方法及力價表示 11 八、蛋白活性力價之比較 11 九、外源蛋白添加之效果 11 （一）蛋白活性力價含量 12 （二）蛋白特性之差異性 13 （三）飼料蛋白質（受質）之不同 13 （四）礦物質之影響 13 （五）動物之生長階段 13 肆、材料與方法 15 Ⅰ. 利用化學分析法評估不同蛋白對飼料蛋白質之作用 15 Ⅱ. 以動物試驗探討外源蛋白對飼料蛋白質於肉雞之消化效果 18 伍、結果與討論 23 Ⅰ. 利用化學分析法評估不同蛋白對飼料蛋白質之作用 23 一、酪胺酸之釋出量 23 二、飼料蛋白質水解後殘留物之氮含量汲水解率 29 三、飼料蛋白質水解後可溶性蛋白質之含量及其分子量分佈 31 四、飼料蛋白質水解產物之蛋白分佈 33 Ⅱ. 以動物試驗探討外源蛋白對飼料蛋白質於肉雞之消化效果 45 一、消化率試驗 45 二、消化道食糜之pH值 47 三、消化道相對重量 51 四、食糜蛋白分佈 54 五、消化道食糜之可溶性蛋白質分子量分佈 62 陸、結論 74 柒、參考文獻 75 捌、英文摘要 87 玖、八十七年中畜年會論文發表（獲新人獎） 89 壹拾、小傳 9

以轉殖植物生產高甲硫胺酸蛋白之方法及其所製得之產品

本發明關於生產高甲硫胺酸蛋白之方法，其係利用植物基因轉殖技術將芝麻種子中2S清蛋白(albumin)基因轉殖於植物中，使該轉殖植物特異地表現富含甲硫胺酸之蛋白質，以提高作物之營養品質。本發明亦關於利用該方法所製得之高甲硫胺酸蛋白，以及包含該蛋白之作物加工產品，其可用作為素食者及食草性動物之營養補給物

Evaluation the potential utilization of feed grade rice for animal usage

一般水稻主要生產作為食用，水稻植株及莖桿、稻穀和米糠等也可以作為動物的食料，事實上取乳熟至黃熟期的稻株可作青貯料作為反芻動物的食料，其營養價值有如一般牧草青貯料。由於稻的種原豐富且具多樣性，本計畫擬篩選具高生產潛力之稻米品種(系)，評估其生產潛、飼料用品質以及總營養消化量，探討其作為飼料用途的潛能，以開發稻米生產的多元化利用。Animals are fed paddy rice and its by-products such as rice straw, rice hull, and rice bran. Whole rice plants can be fed to dairy and beef cattle as whole crop silage. Its nutritional value is almost equivalent to that of barley whole crop silages. This study was carried out to obtain basic information on variety selection and potential performance for the utilization of whole crop rice in Taiwan. Several varieties and elite lines were evaluated on agronomic performance and feed value such as total above-ground dry matter yield, acid detergent fiber, neutral detergent fiber, and total digestible nutrients to meet the potential utilization of rice for whole crop use

人造油體抗原展現系統

本發明係關於一種人造油體抗原展現系統,其特徵在於利用載體蛋白攜帶抗原物質,並鑲嵌於脂溶性顆粒中,而將抗原物質展現於脂溶性顆粒表面,提供作為抗體製備之外來抗原。本發明亦包含該人造油體抗原展現系統之製備方法,以及其用於疫苗製備之用途

於榖類植物榖粒外皮表現外源蛋白質之方法

本發明係關於藉由將外源蛋白質基因以遺傳工程操作與植物(例如水稻)油體膜蛋白基因啟動子(oleosin promoter)連結，而於該特異性啟動子之操控下，使該外源蛋白質專一地表現於穀類植物穀粒外皮(例如稻米之米糠)的蛋白質表現方法。本發明亦包含於穀類植物種子外皮特異表現外源蛋白質之表現載體及其製法，其中該表現載體包含一種植物油體膜蛋白基因啟動子及外源蛋白質基因

於穀類植物榖粒外皮表現外源蛋白質之方法

本發明係關於藉由將外源蛋白質基因以遺傳工程操作與植物(例如水稻)油體膜蛋白基因啟動子(oleosin promoter)連結，而於該特異性啟動子之操控下，使該外源蛋白質專一地表現於穀類植物穀粒外皮(例如稻米之米糠)的蛋白質表現方法。本發明亦包含於穀類植物種子外皮特異表現外源蛋白質之表現載體及其製法，其中該表現載體包含一種植物油體膜蛋白基因啟動子及外源蛋白質基因