基于生物光谱对等离子体诱变雨生红球藻机理及突变体快速筛选方法的研究

发布时间：2018-01-06 12:35

本文关键词：基于生物光谱对等离子体诱变雨生红球藻机理及突变体快速筛选方法的研究　出处：《中国科学技术大学》2016年博士论文　论文类型：学位论文

【摘要】：虾青素作为一种极强的抗氧化剂具有抵御紫外线、增强机体免疫、消炎、抗癌和着色等多种重要的生物学功能,因此在化妆品、医药、保健和水产等领域具有广泛的应用前景。然而虾青素在动物和人体内不能自身合成,需要从体外摄取。雨生红球藻作为一种淡水生的单细胞绿藻是天然虾青素生产的主要来源。然而在规模化养殖中雨生红球藻具有生长速率低、易遭受外源生物的污染和生物量低的缺点,这就需要对原始的出发藻株进行诱变选育。在本论文中我们采用操作简单、诱变效率较高的大气压介质阻挡放电(DBD)对雨生红球藻进行诱变以获得高产虾青素的突变藻株,并对低温等离子体作用于雨生红球藻的诱变效应进行了初步探索。在获得高产有益突变株中,除了应用高效的诱变技术我们还建立能够应用于研究和生产实践的快速、有效的生物光谱快速筛选方法。论文的主要研究成果总结如下：1、低温等离子体诱变实验条件和参数优化。我们利用低温等离子体对雨生红球藻出发株进行诱变处理,通过优化作用剂量、放电时间等参数,确定了最佳的诱变条件并得到了诸多雨生红球藻突变株。2、高产虾青素雨生红球藻光谱筛选的研究。我们建立了基于红外和拉曼显微光谱成像技术对高产虾青素突变藻株的快速筛选方法,并且能够在微观尺度上对虾青素、β胡萝卜素、蛋白质、脂类和多糖的含量和空间分布进行快速定量分析。尤其是在红外光谱中I(1740)/I(1156)与虾青素的含量一致,可以作为反映不同藻株中虾青素含量的一个重要指标。同时,利用拉曼光谱显微成像并结合多元曲线分辨(MCR)分析方法,能够在单细胞水平上对虾青素、p胡萝卜素和叶绿素的含量实现定量分析。此外,应用红外和拉曼光谱结合主成分分析(PCA)能够实现不同虾青素产量的雨生红球藻突变株快速区分。我们还应用近红外光谱(NIRS)技术并结合生化检测建立了基于偏最小二乘(PLS)的生物量、虾青素含量及虾青素在藻细胞的千重的百分含量的预测模型,进一步我们又对不同虾青素产量的雨生红球藻突变株进行预测。3.高产虾青素突变株特性的研究。我们对经过DBD诱变得到的高产虾青素雨生红球藻突变株的生理生化进行了进一步的分析。比如,应用随机扩增多态性DNA (RAPD)对遗传变异进行检测,应用高效液相色谱(HPLC)对抑制剂尼古丁和二苯胺(Nicotine和Diphenylamine)作用下的虾青素合成路径中的各类色素含量进行了分析。同时,我们还分别应用脉冲振幅调制荧光技术(PAMF)和实时定量PCR对不同藻株的光合作用活力和基因表达水平进行了检测。实验结果表明突变株中虾青素合成水平的提高与催化番茄红素到β胡萝卜素转变的番茄红素环化酶(LCY)的基因表达水平的升高有关。4.低温等离子体作用的生物学效应和机理的研究。我们利用生化测定法对等离子作用后的雨生红球藻细胞进行了检测和分析,证实了放电过程中的自由基是引发各类生物学效应的一个重要因素。
[Abstract]:Astaxanthin as a strong antioxidant with ultraviolet resistance, enhancing immunity, anti-inflammatory, anti-cancer and other biological functions and coloring matters, so in cosmetics, medicine, health care and has a wide application prospect. However, aquaculture and other areas of astaxanthin can not be synthesized in animal and human body, need from Haematococcus uptake in vitro. As a kind of freshwater algae of unicellular green algae is the main source of natural astaxanthin production. However, in the moderate scale breeding of haematococcuspluvialis has a lower growth rate, the amount of vulnerable to pollution and biological exogenous biological low, this requires the original starting strain for mutation breeding. In this paper we use simple, atmospheric dielectric barrier discharge induced higher efficiency (DBD) alge haematococcuspluvialis were mutated to obtain high yield of astaxanthin mutant, and the low temperature plasma A preliminary exploration on the mutagenic effects of plovers pluvialis. In high yield beneficial strains, in addition to mutagenesis application we also establish efficient and can be applied to the research and practice of fast and effective method for rapid screening of biological spectrum. The main research results of this paper are summarized as follows: 1, mutagenesis and parameter optimization of experimental conditions low temperature plasma. We use low temperature plasma alge haematococcuspluvialis strain were treated by optimizing the function, dose, discharge time and other parameters to determine the best mutafacient condition and get a lot of haematococcuspluvialis strain.2 Spectra Study on algae screening red rain. We established the astaxanthin producing method for rapid screening of strains the change on the production of astaxanthin infrared and Raman microscopic spectral imaging technology based on, and can at the micro scale of astaxanthin, beta carotene, eggs White matter, the distribution of lipids and polysaccharides in space and rapid quantitative analysis. Especially in the infrared spectrum of I (1740) /I (1156) consistent with the content of astaxanthin, can be used as an indicator to reflect the different strains of the astaxanthin content of algae. At the same time, microscopy and multivariate curve resolution using Raman the spectral analysis method (MCR), can be at the single cell level of astaxanthin, P carotene and chlorophyll content to achieve quantitative analysis. In addition, the application of infrared and Raman spectroscopy combined with principal component (PCA) to achieve different astaxanthin yield of haematococcuspluvialis mutants. We also distinguish the rapid application of near infrared spectroscopy (NIRS) technology combined with biochemical detection was established based on partial least squares (PLS) prediction model in biomass, algal cell dry weight percentage of the astaxanthin content and astaxanthin, further we are on different astaxanthin production The amount of haematococcuspluvialis mutant of.3. mutant of astaxanthin producing characteristics. We obtained by DBD mutagenesis of high H.pluvialis mutant physiology and biochemistry were further analyzed. For example, using random amplified polymorphic DNA (RAPD) was used to detect the genetic variation and application of high performance liquid chromatography chromatography (HPLC) inhibitors of nicotine and two aniline (Nicotine and Diphenylamine) all kinds of pigment content of astaxanthin biosynthesis pathway were analyzed. At the same time, we also used fluorescence pulse amplitude modulation technique (PAMF) and real-time quantitative PCR on photosynthesis activity and gene expression of different strains were detected. The experimental results show that the improved tomato red mutant and catalytic synthesis of astaxanthin levels of the hormone to beta carotene transformation of lycopene cyclase (LCY) gene expression level rise Study on the biological effect and mechanism of high.4. low temperature plasma effect. We use biochemical determination of haematococcuspluvialis after plasma cells were detected and analyzed, confirmed the discharge process of free radicals is an important factor which causes various biological effects.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q949.2

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