激光辐照微藻生物学效应及其诱变育种的研究
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摘要
本文以7种不同门类的微藻为材料,选用波长分布从488nm的蓝光区到1.34μm的远红外区的4种激光,进行激光辐照微藻的生物学效应研究和激光诱变育种的应用尝试。本研究可为激光与藻类细胞相互作用的机理研究提供可靠的实验数据,为激光技术在微藻研究领域的进一步应用提供可资借鉴的方法,也为微藻遗传种质的改良以及优良品系的选育提供新思路、新方法。
首先,利用不同的激光辐照处理不同类型的微藻细胞,探索激光辐照微藻的有效方法,确定有效的激光参数和生物参数。在传统的生理生化测定的基础上,进一步借助电子显微镜、激光共焦扫描显微镜等现代光电技术手段,对激光辐照微藻产生的生物学效应进行比较系统的研究。研究结果表明:激光辐照效果与其波长、功率及辐照剂量有着密切的关系;不同种类的微藻细胞对激光辐照的耐受能力有所不同;微藻细胞的生理状态也影响激光辐照的效果。一定范围的低剂量激光辐照,可促进藻细胞的增殖生长,并提高细胞生物量和代谢产物的含量。具促长效果的藻细胞,线粒体数量有效增加,色素体内的光合片层进一步发育,细胞外的覆被层得以加强;自体荧光光谱无明显的荧光峰位变化,但荧光强度有较大的增强;细胞的色素吸收峰也呈现明显的提高。分析结果认为,激光对藻细胞的生理刺激效应,与细胞超微结构的变化,以及光合色素的有效增加有关,从而改善光合作用活性,较高剂量的激光辐照则对藻细胞有致死和诱变作用。
其次,对具高经济价值雨生红球藻和紫球藻进行激光诱发突变和突变株生理生化特性的研究。本实验采用Nd:YAP激光为诱变剂,先采用逐级稀释平板涂布法进行初筛,在获得直径大、颜色深的藻落后,通过生物量、色素、胞外多糖、蛋白质和不饱和脂肪酸等代谢产物的含量测定,以及重要生物酶的活性检测,进一步分析突变株的遗传特性变化,最终获得生长繁殖快速、抗逆性强、目标产物含量高的优良藻株:即红球藻的H-NO.12诱变株和紫球藻的P-NO.5诱变株。研究结果表明:相对于出发株,所获得的诱变株其各项生理指标含量相对出发藻株都有了较大程度的提高,所筛选的雨生红球藻H-NO.12诱变株在生长速率、细胞密度、色素含量和干重都较出发株提高明显。雨生红球藻最重要的产物虾青素含量则较出发株提高46.37%,提高极显著,可视为高产虾青素的优质藻株。所筛选的紫球藻P-NO.5诱变株较出发株生物量、生长速率提高显著,其色素及相关代谢产物也有较大的提高,胞外多糖产量最大提高36.33%;BPE含量平均提高达166.33%;在不饱和脂肪酸含量上,亚油酸、特别是AA和EPA含量绝对值都较出发株有较大的提高,几种重要的抗氧化酶体系的生物酶的活性也有显著提高显著,抗逆境能力增强。同时,由传代稳定性实验结果看出,选育的诱变藻株具有良好的遗传稳定性。可以认为所获得的诱变株为优良藻株。本研究还进一步利用光生物反应器对紫球藻P-NO.5突变株进行规模培养的尝试,通过对细胞生物量和主要代谢产物产量的分析测定,进一步证实P-NO.5株的遗传稳定性,以及规模培养的可行性。
In this paper,the effective method and experiment parameter of laser irradiating seven microalgae was research, using four kinds of laser(from the 488nm wavelength distribution of the Blu-ray Disc 1.34μm area to the far-infrared region) irradiating these microalgae.This research result provides an experiment foundation for the field of laser employing in algae research and experiment base for laser biological effect mechanism research. Meanwhile,the result can provides a new idea or method of the modified algae and the culture of high qulity strains.
Firstly,the influence induced by laser parameter,including dose,wavelength,power and so on.During laser irradiating,these algae was studied by the numbers.the biological effect induced by laser scanning confocal microscope, LSCM, UV/VIS/NIR spectrophotometer and transmission electron microscope, TEM simultaneity.The experiment result show that:1.There were the best dose,the most functional wavelength and the best combination of irradiating time and power in the course of laser irradiating these algae. 2.Different types of microalgae cells had different tolerant to the laser radiation. 3.The physiological state of microalgae cells had also effected the effection of laser irradiation.
A range of low-dose irradiation, can promote the proliferation of algae growth, and improve the cell biomass and the content of metabolic product. The number of chondria of the algal cell with promoting effect increase effectively. The photosynthetic pigment in lamellar development futher, and the cells cover lager to enhance. The self-fluorescence spectroscopy, no significant changes in the fluorescence peaks. But the fluorescence intensity increased obviously. Meanwhile, the absorption peak of the cell pigment also enhanced obviously.
Analysis the results, we know that the stimulus of the physiological and the effects of the laser on the algal cells are closely related to the changes in the the ultrastructure of cells, as well as the effective increase of the photosynthetic pigment. Thereby ,it improved the photosynthesis activity. The higher dose of laser irradiation on the algal cells have the lethal and mutagenic effect.
Secondly, the laser-induced mutation and mutant physiological and biochemical characteristics of two high economic value alga:Haematococcus pluvialis and P.cruentums had been studied. In the experiment, we used Nd:YAP laser as mutagenic agent, the first step was diameter by step dilution plate coated screening method. Then, determ the pigment and the metabolites such as extracllular polysaccharides, proteins, unsaturated fatty acids as well as the activity detection of some important enzyme. Futher analysis, the genetic characteristics changes of mutant. Eventually, we got the fine lines with rapid growth, reproduction, strong resistance and high content of the target product:the H-N0.12 and the P-N0.5 mutation strains.
The results show that:compared to the originals, the mutaion of H-N0.12 strain had a greater degree improvement in the growth rate, cell density, pigment content and dry weight. The content of the most important product—astaxanthin in the mutation strain increased by 46.37 %. It can be regarded as high quality algal strain with high-yielding quality astaxanthin. The P-NO.5 mutation strain also had increased ih this physiological and biochemical indicators. The ectracellular polysaccharide production increased 36.33 %, BPE content increased 166.33%, and the content of linoleic acid, the absolute value of AA and EPA and the enzyme activity improvement significantly. The capability of adversity was increasing. Therefore, it can also be regard as high quality algal strain. Futher, this study used the optical bioreactor to culture the P-NO.5 strain. Then, the cell biomass and the analysis of the major metabolite confirmed that, the genetic strains stability of P-NO.5, as well as the feasilility of scale cultivation.
首先,利用不同的激光辐照处理不同类型的微藻细胞,探索激光辐照微藻的有效方法,确定有效的激光参数和生物参数。在传统的生理生化测定的基础上,进一步借助电子显微镜、激光共焦扫描显微镜等现代光电技术手段,对激光辐照微藻产生的生物学效应进行比较系统的研究。研究结果表明:激光辐照效果与其波长、功率及辐照剂量有着密切的关系;不同种类的微藻细胞对激光辐照的耐受能力有所不同;微藻细胞的生理状态也影响激光辐照的效果。一定范围的低剂量激光辐照,可促进藻细胞的增殖生长,并提高细胞生物量和代谢产物的含量。具促长效果的藻细胞,线粒体数量有效增加,色素体内的光合片层进一步发育,细胞外的覆被层得以加强;自体荧光光谱无明显的荧光峰位变化,但荧光强度有较大的增强;细胞的色素吸收峰也呈现明显的提高。分析结果认为,激光对藻细胞的生理刺激效应,与细胞超微结构的变化,以及光合色素的有效增加有关,从而改善光合作用活性,较高剂量的激光辐照则对藻细胞有致死和诱变作用。
其次,对具高经济价值雨生红球藻和紫球藻进行激光诱发突变和突变株生理生化特性的研究。本实验采用Nd:YAP激光为诱变剂,先采用逐级稀释平板涂布法进行初筛,在获得直径大、颜色深的藻落后,通过生物量、色素、胞外多糖、蛋白质和不饱和脂肪酸等代谢产物的含量测定,以及重要生物酶的活性检测,进一步分析突变株的遗传特性变化,最终获得生长繁殖快速、抗逆性强、目标产物含量高的优良藻株:即红球藻的H-NO.12诱变株和紫球藻的P-NO.5诱变株。研究结果表明:相对于出发株,所获得的诱变株其各项生理指标含量相对出发藻株都有了较大程度的提高,所筛选的雨生红球藻H-NO.12诱变株在生长速率、细胞密度、色素含量和干重都较出发株提高明显。雨生红球藻最重要的产物虾青素含量则较出发株提高46.37%,提高极显著,可视为高产虾青素的优质藻株。所筛选的紫球藻P-NO.5诱变株较出发株生物量、生长速率提高显著,其色素及相关代谢产物也有较大的提高,胞外多糖产量最大提高36.33%;BPE含量平均提高达166.33%;在不饱和脂肪酸含量上,亚油酸、特别是AA和EPA含量绝对值都较出发株有较大的提高,几种重要的抗氧化酶体系的生物酶的活性也有显著提高显著,抗逆境能力增强。同时,由传代稳定性实验结果看出,选育的诱变藻株具有良好的遗传稳定性。可以认为所获得的诱变株为优良藻株。本研究还进一步利用光生物反应器对紫球藻P-NO.5突变株进行规模培养的尝试,通过对细胞生物量和主要代谢产物产量的分析测定,进一步证实P-NO.5株的遗传稳定性,以及规模培养的可行性。
In this paper,the effective method and experiment parameter of laser irradiating seven microalgae was research, using four kinds of laser(from the 488nm wavelength distribution of the Blu-ray Disc 1.34μm area to the far-infrared region) irradiating these microalgae.This research result provides an experiment foundation for the field of laser employing in algae research and experiment base for laser biological effect mechanism research. Meanwhile,the result can provides a new idea or method of the modified algae and the culture of high qulity strains.
Firstly,the influence induced by laser parameter,including dose,wavelength,power and so on.During laser irradiating,these algae was studied by the numbers.the biological effect induced by laser scanning confocal microscope, LSCM, UV/VIS/NIR spectrophotometer and transmission electron microscope, TEM simultaneity.The experiment result show that:1.There were the best dose,the most functional wavelength and the best combination of irradiating time and power in the course of laser irradiating these algae. 2.Different types of microalgae cells had different tolerant to the laser radiation. 3.The physiological state of microalgae cells had also effected the effection of laser irradiation.
A range of low-dose irradiation, can promote the proliferation of algae growth, and improve the cell biomass and the content of metabolic product. The number of chondria of the algal cell with promoting effect increase effectively. The photosynthetic pigment in lamellar development futher, and the cells cover lager to enhance. The self-fluorescence spectroscopy, no significant changes in the fluorescence peaks. But the fluorescence intensity increased obviously. Meanwhile, the absorption peak of the cell pigment also enhanced obviously.
Analysis the results, we know that the stimulus of the physiological and the effects of the laser on the algal cells are closely related to the changes in the the ultrastructure of cells, as well as the effective increase of the photosynthetic pigment. Thereby ,it improved the photosynthesis activity. The higher dose of laser irradiation on the algal cells have the lethal and mutagenic effect.
Secondly, the laser-induced mutation and mutant physiological and biochemical characteristics of two high economic value alga:Haematococcus pluvialis and P.cruentums had been studied. In the experiment, we used Nd:YAP laser as mutagenic agent, the first step was diameter by step dilution plate coated screening method. Then, determ the pigment and the metabolites such as extracllular polysaccharides, proteins, unsaturated fatty acids as well as the activity detection of some important enzyme. Futher analysis, the genetic characteristics changes of mutant. Eventually, we got the fine lines with rapid growth, reproduction, strong resistance and high content of the target product:the H-N0.12 and the P-N0.5 mutation strains.
The results show that:compared to the originals, the mutaion of H-N0.12 strain had a greater degree improvement in the growth rate, cell density, pigment content and dry weight. The content of the most important product—astaxanthin in the mutation strain increased by 46.37 %. It can be regarded as high quality algal strain with high-yielding quality astaxanthin. The P-NO.5 mutation strain also had increased ih this physiological and biochemical indicators. The ectracellular polysaccharide production increased 36.33 %, BPE content increased 166.33%, and the content of linoleic acid, the absolute value of AA and EPA and the enzyme activity improvement significantly. The capability of adversity was increasing. Therefore, it can also be regard as high quality algal strain. Futher, this study used the optical bioreactor to culture the P-NO.5 strain. Then, the cell biomass and the analysis of the major metabolite confirmed that, the genetic strains stability of P-NO.5, as well as the feasilility of scale cultivation.
引文
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