【作者】 陈莉；

【导师】 任继周；

【作者基本信息】 甘肃农业大学 ， 草业科学， 2011， 博士

【摘要】 为了系统全面的研究苜蓿悬浮细胞培养中代谢产物蛋白质的反应动力学,分别从苜蓿品种、外植体、培养条件、愈伤组织和悬浮细胞系等方面进行了优选,对苜蓿细胞生长过程中营养物质的消耗动力学和生长结构动力学模型进行了探讨,为大规模细胞培养生产苜蓿蛋白提供了基础理论研究依据。根据亲缘关系较远的五种苜蓿中叶组织的蛋白酶活和蛋白质含量的测定,选择苜蓿王（WL215）作为苜蓿的供试品种。进行了苜蓿叶愈伤组织的诱导以及高产蛋白苜蓿悬浮细胞系的筛选。通过单因子试验,分别确定在600lx,12h循环光照条件下,采用0.3cmx0.3cm大小,苜蓿叶脉处外植体在MB+2.0mg/L 2,4-D+0.5mg/L 6-BA的优化培养基中可诱导出多量且较疏松的愈伤组织。筛选获得的苜蓿高产蛋白悬浮细胞系培养12d后生长指数及蛋白总酶活均达到最大值,分别为6.94和10.14x104U/L。通过单因素变化考察了培养基中主要营养成分对悬浮培养苜蓿细胞生长和蛋白积累的影响。蔗糖是适合苜蓿细胞悬浮培养的碳源,不仅有利于细胞生长,并有较高的蛋白产量。在1%-6%范围内,苜蓿细胞生物量和蛋白积累量在3%蔗糖浓度下达到最高。MB培养基的基本氮源已经足够满足苜蓿细胞生长和蛋白合成的需要。NH₄⁺和NO₃^-作为无机氮源提供的离子较易被苜蓿细胞吸收利用,尿素和酪蛋白水解物有助于苜蓿细胞的生长。磷酸盐对苜蓿细胞生长和蛋白合成都是必需的。细胞在无磷培养基中不生长,不同起始PO43-浓度对蛋白合成影响较大。1.25mM磷源浓度下苜蓿细胞生长和蛋白的合成最佳。苜蓿细胞生长过程呈现较典型的“S”型曲线。低浓度时蔗糖成为细胞生长的限制性基质;提高蔗糖浓度可明显促进细胞生长,但浓度超过3%后则表现出对生长的底物抑制作用。不同NH₄⁺/NO₃^-比例对苜蓿细胞生长的影响主要体现在高NH₄⁺浓度对细胞生长的抑制作用。低浓度时磷酸盐成为苜蓿细胞生长的限制性因素。提高磷酸盐浓度细胞生长周期缩短。在基准培养条件下,苜蓿细胞的比生长速率约为0.31⁰.34day-1,倍增时间约为3.4³.8天。不同起始蔗糖浓度下蛋白积累的趋势相似,在一定范围内,增加起始蔗糖浓度有利于蛋白的合成,但在高糖浓度下,蛋白总酶活下降。采用单一NH₄⁺为氮源培养时蛋白总酶活最低,其他氮源配比下蛋白的总酶活均可达到一定的水平,当NH₄⁺/NO₃^-为20:40时,蛋白总活力达到最大。初始磷酸盐的浓度对苜蓿细胞蛋白积累影响显著,1倍磷源浓度时蛋白积累可达到最大值。不同培养条件下,蛋白的积累与苜蓿细胞的生长呈相对密切的偶联关系,蛋白的积累与苜蓿细胞生长呈正相关。苜蓿细胞对糖的吸收速率和利用效率以及胞内糖的积累与培养基的组成有很大关系。通过线性回归计算了各种培养条件下苜蓿细胞及蛋白对糖的得率。苜蓿细胞对NH₄⁺和NO₃^-离子的吸收差异较大,NH₄⁺离子在培养初期即被快速吸收,而NO₃^-离子的吸收较缓慢,细胞内NH₄⁺和NO₃^-离子的积累不明显。苜蓿细胞对磷酸盐吸收快速,起始磷源浓度越高,被细胞利用的速率越快。胞内磷酸盐的积累水平与培养基初始磷酸盐浓度有关。苜蓿细胞蛋白含量的积累与胞内磷酸盐的积累水平成反比。胞内磷酸盐的积累水平对苜蓿细胞的生长和蛋白的合成有重要的影响。构建了苜蓿细胞悬浮培养的结构动力学模型,整个培养系统分为生物相和非物相两部分。生物相是苜蓿细胞内部,被分为四个部分:可溶性糖G、游离磷盐P、中间代谢产物M、代谢产物蛋白S;非生物相指苜蓿细胞外培养基,包括蔗糖Se、还原糖Ge和磷酸盐Pe三个部分。模型的模拟结果与测定值基本吻合,能应用于苜蓿细胞培养过程。更多还原

【Abstract】 Induction Alfalfa suspension cell cultures in the kinetics of protein metabolism suspension cell line were studied. That are optimized from Alfalfa varieties, explants, culture conditions, callus and suspension cell lines, etc. The process of cell growth on the nutrition of Alfalfa material consumption and growth kinetics were discussed structural dynamics model for large-scale production of alfalfa protein in cell culture provide a basis for fundamental research.According to the paternity far five kinds of Alfalfa are determinate the protein protease alive and protein content that choice the Alfalfa （WL215）. Callus of the Alfalfa induction and high production protein of Alfalfa lines suspended selection. Selected the optimized of suspension cell culture the explants size with epidemus was 0.3cm X 0.3cm, more loose callus could be induced from the WL215 in the medium of MB+ 3.0 mg/L 2,4-D+0.5mg/L 6-BA under condition of 600lux 12hours circular illumination. The maximum growth indices was 6.49 while the maximum total Protein activity was 10.14x 104U/L selected Alfalfa cell line suspension after 12 days’ culture.The effects of major nutrients on Alfalfa cell growth and protein production were discussed. Sucrose was better for cell growth and protein production among different carbon sources. In the range of 1%⁶% sucrose concentration,highest biomass and protein production were achieved on 3%. Standard nitrogen concentration in MB medium was enough for Alfalfa cell growth and protein production. Mineral nitrate source and urea were suitable for cell growth. Phosphate was necessary for cell growth and protein production. Different initial phosphate concentration had remarkable effect on protein production. The standard phosphate concentration appeared in 1.25mM more suitable for cell growth and protein production.The suspension culture process of Alfalfa cell could be appeared“S”grow curve. Sucrose was growth limiting nutrient on low concentration condition. Increase of sucrose concentration stimulated cell growth, extended rapid growth phase and increased the final cell harvest. Substrate inhibition was observed when the concentration was above 3%. The influence of different NH₄⁺ / N03- ratio on cell growth was mainly reflected by the inhibition of high ammonium. Phosphate was also the growth limiting nutrient on low concentration condition. Increase of phosphate concentration increased cell growth rate and shortened the culture period. Under the standard culture condition, the specific cell growth rate was 0.31⁰.34 day-1, and the doubling time was 3.4³.8 days.The trends of protein production variation under different initial sucrose concentration were the same. Increase of sucrose concentration could also improve protein production to some extent, while higher sucrose concentration inhibited protein production. Protein production was the lowest when used ammonium as only nitrate source. The maximum total protein activity could be achieved while the ratio of NH₄⁺ / N03- was 20/40. Initial phosphate concentration was most effective on protein accumulation; the maximum protein yield could be obtained under the standard phosphate concentration in 1.25mM.Protein accumulation was partially-growth-associated, protein production by Alfalfa cell suspension was regard as synchronism with cell growth. Alfalfa cell rapidly hydrolyzed extra cellular sucrose before adsorb it. The hydrolyzing process could be described using substrate saturation kinetic equation. Glucose was absorbed more preferentially than fructose. The adsorbing rate, utilizing efficiency and intracellular accumulation of sugar were closely related to medium composition.Ammonium was rapidly adsorbed at the early stage. The adsorption of nitrate was much more slowly. The intracellular accumulation of ammonium and nitrate was not obvious.Phosphate was also rapidly adsorbed at the early stage. The higher the initial phosphate concentration was, the more rapid the utilization rate was. The intracellular phosphate accumulation level related to the initial phosphate concentration in the medium. Protein content in Alfalfa cell was decreased with the increasing of intracellular phosphate accumulation. The accumulation level showed strong influence on Alfalfa cell growth and protein production.A structural kinetic model was constructed in this thesis. The whole culture system was separated into biotic phase and abiotic phase. The former was the inside of Alfalfa cell, which was divided into 4 compartments: soluble sugar G, free phosphate P, Middle metabolites M and products S. The abiotic phase was extra cellular environment, which included sucrose Se, reductive sugar Ge, and phosphate Pe. Intracellular phosphate acted as a regulator on the cell growth and protein accumulation. Calculated result coincided well with experimental data. The model could be used for good description of culture process.更多还原