藉由豬胰澱粉酶啟動⼦基因轉殖策略賦予轉基因豬同時表現外源植酸分解酶與纖維素分解酶之可行性

The Feasibility of a Transgenic Strategy for Co-expression of Phytase and Cellulase Transgenes Driven by the Porcine Pancreatic Amylase Promoter in Pigs

林育聖(Yu-Sheng Lin)

指導教授 ： 鄭登貴

共同指導教授 ： 徐濟泰

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00252

摘要

近年全球面臨氣候變遷與能源轉型、短缺，造成國際榖物供需失衡，國內進口大豆、玉米等主要飼料原料價格高漲，養豬產業飼料成本節節上揚，且畜體飼養過程中所產生之排泄物、廢棄物與其所衍生之環境污染更是目前養豬業者務必嚴正面對之環保課題；職是之故，部分研究先進乃嘗試從營養設計之角度，試圖改良動物對營養分之消化吸收，從而降低大量營養物質隨糞便排出之比例，進而得能有效減少飼料浪費與環境污染之目標。
本研究旨在釐清吾等先前已成功產製以豬胰澱粉酶基因啟動子在胰臟中表現外源之植酸分解酶與纖維素分解酶之轉基因豬，是否具備有效提高消化率與降低糞磷之排放能力；試驗之進行包括：1. 完成分析業經成功選殖2,488-bp之豬胰澱粉酶基因啟動子序列特性以及其活性；2. 依據業經建立之轉基因豬，完成分析並確認彼等外源植酸分解酶與纖維素分解酶等之轉殖基因，分別在其體內之組織表現性及其在性腺被傳承之能力；3. 進行表面消化率試驗，裨謀比較彼等非轉基因豬與轉基因豬，分別被飼予對照組日糧（control diet）與/或低磷日糧（low-P diet）時，各受試豬隻對於飼糧中包括乾物質、粗蛋白質、粗纖維、鈣、磷等之利用的差異性。
試驗結果證明：1. 前述業經選殖出之2,488-bp序列片段被與綠色螢光蛋白基因共同構築成報導載體後，確實能在大鼠胰臟腫瘤細胞株（AR-42J cell line）中成功表現出螢光蛋白質，證明該選殖片段確定具備啟動子之活性，且能有效控制下游基因之表現；2. 以聚合酶反應（polymerase chain reaction, PCR）及南方吸漬法（Southern blot）檢測F1世代基因組，證明先前試驗所產製之轉基因豬具備性腺傳承之能力。此外藉由反轉錄聚合酶鏈鎖反應（reverse transcription-PCR）策略分析F1世代之器官及⁄或組織，結果證實包括植酸分解酶與纖維素分解酶兩者之mRNA表現，分別皆具備有組織之專一性，且僅在胰臟組織中被轉錄。進一步藉由西方吸漬（Western blot）分析包括F1與F2世代轉基因豬之胰臟組織及其腸內容物；結果證明彼等源自被轉殖之植酸分解酶與纖維素分解酶等外源基因，其所表現之蛋白質分別確能在胰臟被合成，且經由胰管分泌進入十二指腸中。此外，經活性試驗結果證明，前述兩種蛋白質在胰臟及十二指腸內容物，且分別具備分解植酸與纖維素之活性，並以十二指腸內容物活性較高；植酸分解酶活性經過鉬藍法（molybdate-blue method）定量計算結果，證明其在十二指腸中存在，且高達到8.2 U/mL之譜；3. 在以玉米、大豆及大麥為主之對照組日糧中，相較於彼等非轉基因豬而言，轉基因豬在乾物質、中性洗滌纖維、粗蛋白質、鈣、及磷等之表面消化率，分別均有較佳之表現，特別是乾物質及中性洗滌纖維等，更呈現有顯著性之差異（P <0.05）；就飼予玉米、大豆及大麥等為主之低磷組日糧者而言，轉基因豬在乾物質、中性洗滌纖維、粗蛋白質、鈣、及磷等之表面消化率，分別均較彼等非基因轉殖豬者，有較佳的表現（P <0.05）。其屬有趣者，乃非基因轉殖豬比較之被飼予對照組及/或低磷組日糧者，並進一步分析其糞便中之氮及磷含量者，試驗結果顯示轉基因豬之被餵飼對照組與低磷組日糧者，其糞便中磷之排出量分別被減少29%和48%之譜，此外其糞便中氮排出量亦分別下降多達31%和49%之譜。
綜合上述試驗結果，本研究證明，試驗所構築之2,488-bp豬胰澱粉酶基因啟動子序列不僅具有啟動轉錄之能力，且在先前產製之轉基因豬所攜帶之外源基因，確定可經由性腺被傳承至其子代。此外，彼等在胰臟組織專一表現植酸分解酶及纖維素分解酶之轉殖基因，其表現之外源轉基因之蛋白質，且分別具備有分解酶之活性，遂能有效增進其對飼料之消化效率從而顯著減少糞中氮及磷之排出量。未來之試驗研究冀望得能進一步完成豬隻在各不同生長階段之相關營養試驗，並針對此等轉基因豬隻各階段之生長速率，對於各不同飼料原料之消化能力，及豬隻在腸道中被免疫相關基因之調控等，可能分別扮演有重要之角色；深盼透過本轉基因殖豬模式之完成建立，不僅得能賦予豬隻具備有效利用高纖維原料替代物（fiber-rich by products）之固有營養分，且能有效維持豬隻生長與繁殖性能，從而更能兼具臻於環保目標之要求。

關鍵字

轉基因豬 ； 豬胰澱粉酶基因啟動子 ； 植酸分解酶 ； 纖維素分解酶

並列摘要

Competition between humans and livestock for cereal and legume grains makes it challenging to provide economical feeds to livestock animals. Recent increases in both corn and soybean prices have had a significant impact on the cost of feed for pig producers. The utilization of by-products and alternative ingredients in pig diets has the potential to reduce feed costs. However, unlike ruminants, pigs have limited ability to utilize diets with high fiber content because they lack endogenous enzymes capable of breaking down non-starch polysaccharides into simple sugars.
In addition, pigs do not produce sufficient endogenous phytase to hydrolyze P from phytate in cereals, making it necessary to supplement with inorganic phosphorus (P) to meet the requirements for optimum pig production, resulting in higher fecal concentrations of P and increasing potential environmental pollution. Thus, increased regulatory pressures are being imposed on animal agriculture to minimize P and nitrogen emissions. Improvement of the digestive efficiency of pigs is the most logical approach for reducing nutrient excretion and achieving pollution control. This study investigated a biological method for reducing fecal P excretion and improving nutrient digestibility, and utilized a transgenic strategy in which expression of bacterial phytase and fungal cellulase in the gastrointestinal tract to establish an eco-friendly pig model.
A 2,488-bp 5′-flanking region of the porcine pancreatic amylase gene was cloned and its structural features were characterized. Using green fluorescent protein as a reporter, we found that this region contained promoter activity and had the potential to control heterologous gene expression. Germ-line transmission and tissue-specific expression of phytase and cellulase transgenes in the pancreas of F1 and F2 transgenic pigs were identified. Both enzyme activities were also detectable in pancreas and duodenal contents. Up to 8.233 U/mL phytase activity was detected in the duodenal contents of transgenic pigs, which represents 5−8 times the activity in non-transgenic pigs. On a corn, barley, and soybean-meal-based control diet, the apparent fecal digestibility of dry matter (DM), neutral detergent fiber (NDF), crude protein, calcium, and P was higher in transgenic pigs than in non-transgenic pigs (P < 0.05 for both DM and NDF digestibility). On a low-P basal diet, the transgenic pigs exhibited significantly increased digestibility of these nutrients (P < 0.05).
Conclusions come to the above studies indicated that those transgenic pigs fed with the control and/or the low-P diet(s) would result in significantly decrease of their fecal P outputs by 29% and 48%, respectively, and also their fecal N outputs was reduced by 31% and 49%, respectively, when comparisons were made to the fecal P and/or N outputs from those non-transgenic pigs. Based on evidences demonstrated above, the establishment of a tissue-specific promoter of the porcine pancreatic amylase gene and suggests our transgenic pigs may represent a way to increase nutrient utilization and reduce fecal P and N contents.

並列關鍵字

transgenic pig ； pancreatic amylase promoter ； phytase ； cellulase

參考文獻

1. Abello, J., T. Corring, and J. P. Laplace. 1987. Contribution of bile and pancreatic juice to the control of pH in the pig duodenum. Reprod. Nutr. Dev. 27:681–687.

2. Abioye, S., D. V. Ige, O. O. Akinremi, D. N. Flaten, and C. M. Nyachoti 2010. Characterizing fecal and manure phosphorus from pigs fed phytase supplemented diets. J. Agric. Sci. 2:3–12.

3. Adeola O. 1999. Nutrient management procedures to enhance environmental conditions: An introduction. J. Anim. Sci. 77:427–429.

5. Adeola, O., J. S. Sands, P. H. Simmins, and H. Schulze. 2004. The efficacy of an Escherichia coli-derived phytase preparation. J. Anim. Sci. 82:2657–2666.

6. AOAC. 1984. Official methods of analysis of the Association of Official Analytical Chemists, 14th edition. Assoc. Offic. Anal. Chem., Arlington, VA.