本文關(guān)鍵詞：赤霉菌Gibberella sp.F75來源的α-半乳糖苷酶轉(zhuǎn)基因玉米的培育　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： α-半乳糖苷酶 轉(zhuǎn)基因玉米 飼用酶制劑 Gibberella sp.F75

【摘要】：豆粕是動(dòng)物飼料中主要的蛋白原料。但是大多數(shù)豆類植物中存在一種α-半乳糖苷鍵連接形成的寡糖,在單胃動(dòng)物的消化系統(tǒng)中降解時(shí)會(huì)伴隨產(chǎn)生大量氣體,致使動(dòng)物出現(xiàn)脹氣和腹瀉等不良癥狀。將α-半乳糖苷酶添加到飼料中水解這些寡糖,可以有效避免上述不良癥狀。雖然微生物發(fā)酵表達(dá)α-半乳糖苷酶具有靈活方便的特點(diǎn),但是該過程需要大量的設(shè)備投資和能源消耗。相對而言,在飼用作物中表達(dá)α-半乳糖苷酶既可以減少環(huán)境污染,節(jié)約能源,又能簡化酶制劑的生產(chǎn)添加過程。玉米是動(dòng)物飼料的另一種主要原料,已經(jīng)被廣泛研究,是理想的生產(chǎn)飼用酶的生物反應(yīng)器。本研究的目的是培育一種能夠高效表達(dá)α-半乳糖苷酶且穩(wěn)定遺傳的轉(zhuǎn)基因玉米品系,直接用于飼料生產(chǎn)。本研究選取來源于赤霉菌Gibberella sp.F75的α-半乳糖苷酶基因aga-F75為目的基因,該基因編碼的酶蛋白具有良好的酶學(xué)性質(zhì)和蛋白酶抗性,具有較高的應(yīng)用價(jià)值。為使aga-F75在玉米中成功表達(dá),對其進(jìn)行了密碼子優(yōu)化改造。優(yōu)化后的α-半乳糖苷酶基因aga-F75m與原基因的核苷酸序列一致性為77.3%,密碼子適應(yīng)指數(shù)CAI值從0.71上升到0.82,GC含量從51.29%提高至55.82%。將aga-F75m連接到含有玉米種子特異性表達(dá)啟動(dòng)子的植物表達(dá)載體pHP20754上,獲得外源基因的植物表達(dá)盒。通過基因槍轉(zhuǎn)化法將植物表達(dá)盒導(dǎo)入玉米Hi-II受體材料中,獲得33個(gè)轉(zhuǎn)化事件。經(jīng)過篩選、分化、生根和成苗最終獲得T0代轉(zhuǎn)基因玉米。選擇優(yōu)良玉米品種鄭58自交系作為父本與轉(zhuǎn)基因玉米雜交傳代,后代再與鄭58進(jìn)行多代回交選育。轉(zhuǎn)基因和非轉(zhuǎn)基因玉米的農(nóng)藝性狀差異分析表明外源基因?qū)D(zhuǎn)基因玉米的表觀性狀沒有明顯影響。對各代轉(zhuǎn)基因玉米種子的α-半乳糖苷酶活性進(jìn)行分析,結(jié)果顯示其種子活性高達(dá)10 000 U/kg,平均酶活力為3 500 U/kg。Southern blot分析表明aga-F75m在轉(zhuǎn)化事件8-1的轉(zhuǎn)基因玉米基因組中至少有兩個(gè)拷貝。Western blot分析顯示酶蛋白Aga-F75M的分子量(95 kDa)大于其理論分子量(82 kDa),而且只在轉(zhuǎn)基因玉米種子中特異性表達(dá),在玉米的根、莖、葉中均未表達(dá)。Aga-F75M經(jīng)糖苷酶PNGase F去糖基化處理后蛋白質(zhì)分子量降低,說明該蛋白存在N-糖基化修飾。與畢赤酵母重組表達(dá)的Aga-F75相比,轉(zhuǎn)基因玉米表達(dá)的Aga-F75M的最適溫度(50°C)降低了10°C,但在最適pH、酸堿穩(wěn)定性和熱穩(wěn)定性方面沒有明顯差異。80°C飼料制粒實(shí)驗(yàn)表明玉米表達(dá)的Aga-F75M的酶活損失率(80%)低于酵母表達(dá)的Aga-F75(100%),說明玉米表達(dá)的α-半乳糖苷酶有更好的制粒穩(wěn)定性。本研究首次將外源α-半乳糖苷酶基因在玉米中成功表達(dá),獲得了植株性狀良好、外源基因成功表達(dá)并穩(wěn)定遺傳的α-半乳糖苷酶轉(zhuǎn)基因玉米。外源基因aga-F75m在轉(zhuǎn)基因玉米中成功表達(dá)充分證實(shí)了將大分子的飼用酶在轉(zhuǎn)基因作物中表達(dá)的可行性。α-半乳糖苷酶轉(zhuǎn)基因玉米作為原料可以直接用于飼料生產(chǎn),不僅免去了酶制劑的發(fā)酵、純化和添加,還減少了能源浪費(fèi)和環(huán)境污染,具有很好的應(yīng)用前景和一定的生態(tài)環(huán)境效益。
[Abstract]:Soybean meal is the main raw material protein in animal feed. But there are linked oligosaccharides form a alpha galactosidase bond most legumes, degradation in the digestive system in monogastric animal will be accompanied by a large number of gases, resulting in the emergence of animal flatulence and diarrhea and other symptoms. The alpha galactosidase is added to feed hydrolysis of these oligosaccharides, can effectively avoid the adverse symptoms. Although microbial fermentation and expression of alpha galactosidase has the characteristics of flexible and convenient, but the process requires a lot of equipment investment and energy consumption. In contrast, as alpha galactosidase can reduce the environmental pollution, the expression in the feed to save energy, and to simplify production of enzyme preparation process. Add corn is another main raw material of animal feed, has been widely studied, is a bioreactor to produce enzyme ideal. The purpose of this study is to To cultivate a high expression of alpha galactosidase and genetic stability of transgenic maize, directly used for feed production. This study selected from Gibberella sp.F75 Gibberella alpha galactosidase gene aga-F75 gene, the gene encoding the enzyme protein with enzymatic properties of protease resistant and good application value. High. The successful expression of aga-F75 in maize, the optimization of the codon. The nucleotide sequence consistency after optimization of alpha galactosidase gene and aga-F75m gene 77.3% codon adaptation index CAI value increased from 0.71 to 0.82, the content of GC increased from 51.29% to 55.82%. aga-F75m connected to the containing corn seed specific expression promoter and plant expression vector pHP20754, obtained the exogenous gene expression in plant. The plant expression box box into maize H by Biolistic Method I-II receptor material, obtained 33 transformation events. After screening, differentiation, rooting and seedling obtained T0 transgenic maize. Maize varieties Zheng 58 inbred lines as male parent and transgenic maize hybrid progenies were passaged, multi backcross breeding and Zheng 58. Analysis of transgenic and non transgenic maize agronomic traits difference showed that the foreign gene of transgenic maize on apparent properties have no obvious influence on the enzyme activity of the transgenic maize seeds of alpha galactose were analyzed, the results showed that the seed activity up to 10000 U/kg, the average enzyme activity was 3500 U/kg.Southern blot analysis showed that in the event 8-1 transgenic maize genome aga-F75m in at least two copies.Western blot analysis showed that the enzyme protein molecular weight of Aga-F75M (95 kDa) is larger than the theoretical molecular weight (82 kDa), and only in transgenic corn seed specificity The expression in maize roots, stems and leaves were not the expression of protein.Aga-F75M by glucosidase PNGase F deglycosylation treatment decreased, indicating that the presence of N- protein glycosylation. Compared with expression in Pichia pastoris Aga-F75, the optimum temperature of transgenic maize expressing Aga-F75M (50 ~ C) decreased by 10 ~ C, but in the most suitable pH, pH stability and thermal stability and there is no significant difference in.80 ~ C feed pelleting experiments showed that maize expression of Aga-F75M enzyme activity loss rate (80%) lower than the yeast expression of Aga-F75 (100%), indicating the expression of alpha granule stability of corn galactose glucoside enzyme is better for the first time in this study. The successful expression of exogenous alpha galactosidase gene in maize, obtained good plant traits, exogenous gene was successfully expressed and the genetic stability of alpha galactosidase transgenic maize. The successful expression of aga-F75m gene in transgenic maize Fully demonstrated the feasibility of the enzyme molecule expression in transgenic crops. Alpha galactosidase transgenic corn as the raw material can be directly used for feed production, not only from the fermentation of enzyme preparation, purification and added, also reduces the waste of energy and environmental pollution, and has good application prospects and the ecological environment benefit.

【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S513;Q943.2

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