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基于原生质体的雨生红球藻转化方法研究
Alternative Title生物工程
程天佑
Thesis Advisor雨生红球藻 ; 差异培养与再生 ; 电转化 ; 基因枪转化 ; 原生质体转化
2017-05
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline雨生红球藻(Haematococcus pluvialis)被公认为是天然虾青素的最佳生物来源之一,但在工业化生产应用中仍存在生物量积累慢、生产周期长、代谢难以调控、虾青素提取成本高等制约因素。而利用基因工程手段对雨生红球藻进行性状改良是特别有效可行的措施。因此本论文以建立操作简单、转化效率高、可稳定遗传的雨生红球藻转化体系,为雨生红球藻藻株改良、虾青素代谢改造等提供方法学以及技术工具基础,并以对雨生红球藻进行营养代谢改造为目标,分析了雨生红球藻游动细胞与不动细胞的分化和差异性培养与再生,并对游动细胞和不动细胞进行电转化尝试与基因枪转化,优化了基于雨生红球藻游动细胞的原生质体制备条件并首次建立雨生红球藻原生质体转化方法。 针对雨生红球藻复杂生活史中主要存在游动细胞和不动细胞两种形态,首先通过观测培养过程的生长与分化,确定这两种形态的细胞组成变化特点,考察了这两种形态的细胞对缓冲液的选择性以及在平板上的再生效率,从而确定了获得单一类型或者单一类型占绝大多数的差异培养方法。实验结果表明通过在BG11中添加乙酸钠和酵母提取物的BYA培养基培养4天后游动细胞超过80%,培养12天后不动细胞超过80%,细胞密度最高达到8×105个/mL,且500 g离心5 min适合于收集所有类型的藻细胞。而在Tris-HCl缓冲液中相比于不动细胞的稳定性,游动细胞的稳定存在需要至少0.2 mmol/L的Ca2+。同时,低熔点琼脂平板再生方式可显著提高雨生红球藻的平板再生效率,而玉米淀粉包裹法的平板再生效率与普通涂布法没有显著差异。进一步的实验结果表明,游动细胞的平板再生效率显著超过不动细胞的平板再生效率,且藻细胞在加入了乙酸钠的TAP和BYA培养基上克隆形成速度超过另两种自养培养基8P-BG11和3N-BBM,藻细胞在BYA培养基上再生效果最好,游动细胞和不动细胞在BYA培养基的再生效率分别可达52.8%、31.5%。研究结果为适合雨生红球藻分子生物学操作的单一形态细胞的获得奠定了基础。 其次,本文分析了雨生红球藻在固体平板以及液体培养基中对壮观霉素和腐草霉素的抗性,并对雨生红球藻的游动细胞和不动细胞进行了电转化和基因枪转化。实验结果表明雨生红球藻在固体平板和液体培养基中对壮观霉素和腐草霉素的敏感浓度差异很大,其在固体环境下的适宜筛选浓度分别为200 µg/mL、8 µg/mL,在液体环境下的适宜筛选浓度分别为20 µg/mL、1 µg/mL。 雨生红球藻游动细胞比不动细胞对电场更敏感,前者在方波场强为1000 V/cm时,细胞存活率约为40%,后者在2000 V/cm时细胞存活率约为40%,但实验重复性较差,多次筛选未得到转化子。因此对藻细胞进行电转化可能需要更进一步的优化。基因枪转化中,通过腐草霉素抗性基因Ble未能筛选到转化子,而通过壮观霉素抗性基因addA筛选到转化子,且通过PCR验证表明通过pHpluS1和18s-pHpluS1-28s质粒同源整合,addA基因分别插入到了雨生红球藻的叶绿体(与已发表的结果类似)和染色体中。 为了进一步丰富雨生红球藻转化方法,基于实验室前期原生质体制备技术,对其制备以及再生条件进行了优化,并基于原生质体首次建立雨生红球藻原生质体转化方法。获得了以下的实验结果:在原生质体制备缓冲液中加入0.5 mmol/L的CaCl2,可显著性地提高原生质体存活率;以酶法处理游动细胞制备原生质体时,蛋白酶K制备效果最好,细胞密度为5×105个/mL时,在35C、 100 r/min的摇床中处理120 min制备效率与存活率达到最佳,分别为78.5%与78.4%。原生质体比游动细胞对电场更敏感,通过电转化筛选平板上首次出现了转化子,但转化子无法长大。建立了PEG介导的eGFP转化,观察到了荧光现象,转化线性化后的pHpluS1质粒与18s-pHpluS1-28s质粒都成功筛选到了转化子。通过PCR验证表明addA基因分别插入到了雨生红球藻叶绿体和染色体中,且在60天内稳定,从而首次成功建立了雨生红球藻原生质体稳定遗传转化体系。
Keyword工学
Abstract中文
Other AbstractHaematococcus pluvialis (Chlorohyceae) is generally regarded as one of the best biological source for astaxanthin which is widely used for health product, feed and medicine. However, the Haematococcus pluvialis through autotrophy is poor in biomass producty and costive when inudstrial production. Genetic engineering modification such as the construction of heterophic strains with glucose may solve the problems. This thesis had made an attempt to establish stable genetic transformation of Haematococcus pluvialis. Research work includes the analysis of the differentiation between motile cell and non-motile cell, the difference in cultivation and regeneration on solid plate for electrotransformation, biolistic transformation and PEG-mediated protoplast transformation. Firstly, based on the morphology of two main cell type - motile cell and non-motile cell in its complicated life history, the cell differentiation of all Haematococcus pluvialis cells to be single type or the majority of cells to be single type during the cultivation were observed. Results showed that motile cell occupied more than 80% in the first 4 days and non-motile cell occupied more than 80% when cultivated for 12 days in BYA medium which contains 2 g/L sodium acetate, 2 g/L yeast extration in BG11. Meanwhile the maximm cells density was 8×105 cells/mL. Under the centrifugation condition of 500 g × 5 min , all types of cells will be collected. In Tris-HCl buffer with 0.2 mmol/L CaCl2, motile cells were more stable while non-motile cells did without CaCl2. In addition, compared with direct coating for regeneration, double-layer plate was proved to be significantly more effective while starch embedding showed no advantage. Futher experiments indicates that the regeneration rate of motile cell was higher than non-motile cell, and colonies in TAP and BYA in which sodium acetate was added formated more quickly than 8P-BG11 and 3N-BBM. BYA was the best medium for cell regeneration and the regeneration rate was 52.8% and 31.5% for motile cell and non-motile cell respectively. Secondly, the sensibility to spectinomycin and zeomycin of Haematococcus pluvialis in solid plates and liuqid medium was analyzed, and electrotransformation and biolistic transformation were conducted. The results suggested that appropriate screening concentration of spectinomycin and zeomycin to Haematococcus pluvialis was 200 µg/mL, 8 µg/mL in solid palte respectively while 20 µg/mL, 1 µg/mL respectively in liquid medimu. Motile cells were more sensitive than non-motile cell to electricity, and the survival rate was 40% when the voltage was about 1000 v/cm for motile cell, but for non-motile cell, the voltage was 2000 v/cm to reach the same survial rate. Unofortunately, the experimental repeatability was poor and recombinant transformant was not observed via Ble gene to zeomycin, however, transformant was found via addA gene to spectinomycin. PCR analysis had proved that the transgene was integrated into the chloroplast genome and chromosome repestively by pHpluS1 plasmid and 18s-pHpluS1-28s plasmid. Finally, based on the optimized protocol for protoplast preparation and regeneration, protoplast transformatioin of Haematococcus pluvialis was constructed for the first time. Protoplast viability was significantly increased when 0.5 mmol/L CaCl2 was added into the buffer solution. Protease-k was proved to be the most effective enzyme for protoplast preparation, and when the cell density was 5×106 cells/mL, preparation rate and viability was 78.5% and 78.4% respectively at 35C for 120 min. Transformant was found by electrotransformation based on protoplast, but the colonies also could’t grow in liquid medium. With PEG (polyethylene glycol) -mediated protoplast transformation, fluorescence was observed. Besides, transformant were found via addA gene to spectinomycin, and it is confirmed that the transgene was integrated into the chloroplast genome and chromosome repestively by pHpluS1 plasmid and 18s-pHpluS1-28s plasmid which were linearized by PCR analysis.
Department微藻生物技术团队
Date Available2021
Subtype硕士 ; 学位论文
Language中文
Document Type学位论文
Identifierhttp://ir.qibebt.ac.cn/handle/337004/9971
Collection微藻生物技术团队
Affiliation中国科学院大学
Recommended Citation
GB/T 7714
程天佑. 基于原生质体的雨生红球藻转化方法研究[D]. 北京. 中国科学院大学,2017.
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