赤霉菌Gibberella sp.F75来源的α-半乳糖苷酶转基因玉米的培育

发布时间：2018-01-09 13:32

本文关键词：赤霉菌Gibberella sp.F75来源的α-半乳糖苷酶转基因玉米的培育　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：豆粕是动物饲料中主要的蛋白原料。但是大多数豆类植物中存在一种α-半乳糖苷键连接形成的寡糖,在单胃动物的消化系统中降解时会伴随产生大量气体,致使动物出现胀气和腹泻等不良症状。将α-半乳糖苷酶添加到饲料中水解这些寡糖,可以有效避免上述不良症状。虽然微生物发酵表达α-半乳糖苷酶具有灵活方便的特点,但是该过程需要大量的设备投资和能源消耗。相对而言,在饲用作物中表达α-半乳糖苷酶既可以减少环境污染,节约能源,又能简化酶制剂的生产添加过程。玉米是动物饲料的另一种主要原料,已经被广泛研究,是理想的生产饲用酶的生物反应器。本研究的目的是培育一种能够高效表达α-半乳糖苷酶且稳定遗传的转基因玉米品系,直接用于饲料生产。本研究选取来源于赤霉菌Gibberella sp.F75的α-半乳糖苷酶基因aga-F75为目的基因,该基因编码的酶蛋白具有良好的酶学性质和蛋白酶抗性,具有较高的应用价值。为使aga-F75在玉米中成功表达,对其进行了密码子优化改造。优化后的α-半乳糖苷酶基因aga-F75m与原基因的核苷酸序列一致性为77.3%,密码子适应指数CAI值从0.71上升到0.82,GC含量从51.29%提高至55.82%。将aga-F75m连接到含有玉米种子特异性表达启动子的植物表达载体pHP20754上,获得外源基因的植物表达盒。通过基因枪转化法将植物表达盒导入玉米Hi-II受体材料中,获得33个转化事件。经过筛选、分化、生根和成苗最终获得T0代转基因玉米。选择优良玉米品种郑58自交系作为父本与转基因玉米杂交传代,后代再与郑58进行多代回交选育。转基因和非转基因玉米的农艺性状差异分析表明外源基因对转基因玉米的表观性状没有明显影响。对各代转基因玉米种子的α-半乳糖苷酶活性进行分析,结果显示其种子活性高达10 000 U/kg,平均酶活力为3 500 U/kg。Southern blot分析表明aga-F75m在转化事件8-1的转基因玉米基因组中至少有两个拷贝。Western blot分析显示酶蛋白Aga-F75M的分子量(95 kDa)大于其理论分子量(82 kDa),而且只在转基因玉米种子中特异性表达,在玉米的根、茎、叶中均未表达。Aga-F75M经糖苷酶PNGase F去糖基化处理后蛋白质分子量降低,说明该蛋白存在N-糖基化修饰。与毕赤酵母重组表达的Aga-F75相比,转基因玉米表达的Aga-F75M的最适温度(50°C)降低了10°C,但在最适pH、酸碱稳定性和热稳定性方面没有明显差异。80°C饲料制粒实验表明玉米表达的Aga-F75M的酶活损失率(80%)低于酵母表达的Aga-F75(100%),说明玉米表达的α-半乳糖苷酶有更好的制粒稳定性。本研究首次将外源α-半乳糖苷酶基因在玉米中成功表达,获得了植株性状良好、外源基因成功表达并稳定遗传的α-半乳糖苷酶转基因玉米。外源基因aga-F75m在转基因玉米中成功表达充分证实了将大分子的饲用酶在转基因作物中表达的可行性。α-半乳糖苷酶转基因玉米作为原料可以直接用于饲料生产,不仅免去了酶制剂的发酵、纯化和添加,还减少了能源浪费和环境污染,具有很好的应用前景和一定的生态环境效益。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S513;Q943.2

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