QIBEBT-IR研究单元&专题: 微藻生物技术研究组http://ir.qibebt.ac.cn:80/handle/0/1382024-03-29T09:26:56Z2024-03-29T09:26:56Z橘色藻适应环境干湿交替的生理机制研究彭宇http://ir.qibebt.ac.cn:80/handle/337004/136592019-12-31T10:57:05Z2019-12-31T10:57:04Z题名: 橘色藻适应环境干湿交替的生理机制研究
作者: 彭宇2019-12-31T10:57:04Z雨生红球藻游动细胞的培养方法及原生质体的制备方法徐晓莹程天佑张维陈林刘天中http://ir.qibebt.ac.cn:80/handle/337004/135832019-12-31T10:39:57Z2019-12-31T10:39:55Z题名: 雨生红球藻游动细胞的培养方法及原生质体的制备方法
作者: 徐晓莹; 程天佑; 张维; 陈林; 刘天中2019-12-31T10:39:55Z一种用于微藻采收的便携式低能耗气浮装置及使用方法刘天中周文俊陈林高莉丽王俊峰汪辉张维程文涛http://ir.qibebt.ac.cn:80/handle/337004/135822019-12-31T10:39:55Z2019-12-31T10:39:53Z题名: 一种用于微藻采收的便携式低能耗气浮装置及使用方法
作者: 刘天中; 周文俊; 陈林; 高莉丽; 王俊峰; 汪辉; 张维; 程文涛2019-12-31T10:39:53Z一种雨生红球藻细胞周期同步化的调控方法及其应用刘天中陈林王俊峰张维汪辉高莉丽周文俊张兰兰http://ir.qibebt.ac.cn:80/handle/337004/135812019-12-31T10:39:53Z2019-12-31T10:39:51Z题名: 一种雨生红球藻细胞周期同步化的调控方法及其应用
作者: 刘天中; 陈林; 王俊峰; 张维; 汪辉; 高莉丽; 周文俊; 张兰兰2019-12-31T10:39:51Z黄丝藻自养和异养模式下生理生化与代谢物组学研究邵慧敏http://ir.qibebt.ac.cn:80/handle/337004/134642018-12-29T22:02:42Z2018-12-29T09:33:05Z题名: 黄丝藻自养和异养模式下生理生化与代谢物组学研究
作者: 邵慧敏2018-12-29T09:33:05Z基于原生质体的雨生红球藻转化方法研究程天佑http://ir.qibebt.ac.cn:80/handle/337004/99712018-08-24T00:58:49Z2018-01-08T10:28:50Z题名: 基于原生质体的雨生红球藻转化方法研究
作者: 程天佑
摘要: Haematococcus 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 35C 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.
摘要: 中文2018-01-08T10:28:50Z周期性间歇光照对栅藻贴壁生长的影响及其反应器的光照特性Andrea Efrem Toninellihttp://ir.qibebt.ac.cn:80/handle/337004/99502018-08-27T22:59:34Z2018-01-08T10:00:49Z题名: 周期性间歇光照对栅藻贴壁生长的影响及其反应器的光照特性
作者: Andrea Efrem Toninelli2018-01-08T10:00:49Z雨生红球藻细胞形态转换和藻落形成差异研究程天佑;徐晓莹;张文蕾;张维; 陈林; 袁冠华; 刘天中http://ir.qibebt.ac.cn:80/handle/337004/98852018-08-27T23:01:22Z2018-01-05T14:18:32Z题名: 雨生红球藻细胞形态转换和藻落形成差异研究
作者: 程天佑;徐晓莹;张文蕾;张维; 陈林; 袁冠华; 刘天中2018-01-05T14:18:32ZHeterotrophy of filamentous oleaginous microalgae Tribonema minus for potential production of lipid and palmitoleic acidWenjun ZhouHui WangLin ChenWentao ChengTianzhong Liuhttp://ir.qibebt.ac.cn:80/handle/337004/98842018-08-27T23:01:23Z2018-01-05T14:18:31Z题名: Heterotrophy of filamentous oleaginous microalgae Tribonema minus for potential production of lipid and palmitoleic acid
作者: Wenjun Zhou; Hui Wang; Lin Chen; Wentao Cheng; Tianzhong Liu2018-01-05T14:18:31Z雨生红球藻原生质体制备及细胞融合研究徐晓莹http://ir.qibebt.ac.cn:80/handle/337004/97832018-08-27T23:02:57Z2017-12-29T12:01:15Z题名: 雨生红球藻原生质体制备及细胞融合研究
作者: 徐晓莹
摘要: Haematococcus pluvialis is considered as the best bioresource in nature to produce astaxanthin. However, the low growth rate and complex culture conditions greatly limit the cultivation efficiency of H. pluvialis. In contrast, Chlorella has strong environmental resistance, fast growth rate and be able to use glucose and other substrates for high-density heterotrophic fermentation. If the new Haematococcus strain that has the ability of heterotrophic growth efficiently on sugar and accumulation of astaxanthin could be obtained through cell fusion, the astaxanthin production efficiency from H. pluvialis would be greatly improved. Therefore, in order to achieve cell fusion of Chlorella and H. pluvialis as well as the screening of sugar heterotrophic mutants, this study established Haematococcus protoplast preparation technology and analyzed the controlled factors of Haematococcus cell differentiation, and finally obtained and identified the fusants successfully.
Firstly, the various cell types in Haematococcus life cycle have a significant impact on protoplast preparation, thus different enzyme treatment conditions should be applied for different cell types. Comparing with Haematococcus non-motile cells, motile cells lack the cellulose cell wall and the protoplast is only surrounded by the extracellular matrix (ECM), which makes motile cells a high osmotic pressure sensitivity. Cell blast under low osmotic pressure is a key indicator to judge the protoplast preparation efficiency, thus the high proportion motile cells in cultivation is often mistaken for obtaining high protoplast preparation rate. Therefore, using the enriched motile cells and non-motile cells, this study compared different enzyme treatment methods and established Haematococcus protoplast preparation technology. Results showed that the ECM is removed from motile cells and completely naked protoplasts are obtained after protease treatment. Collagenase IV is proved to be the most effective enzyme for motile cell protoplast preparation, ECM removal rate is 74.43% and the cell viability is 69.16% by 0.4% collagenase IV degradation at 35℃ for 45 min. In contrast, proteinase K and snailase+cellulase are proved to be the best choice for non-motile cell protoplast preparation, and the highest osmotically-labile cell percentage is about 40%. However, microscopic observation found the cellulosic component is degraded effectively, but the secondary wall (SW) remains intact, which limit the further increase of protoplast preparation efficiency.
Secondly, based on the different sensitivity to Triton X-100 between motile cells and non-motile cells, this study established a novel method for rapid identify cell differentiation during cultivation. The method was applied to compare and analyze the cell growth and differentiation in the medium of BG11, BG11 + ACE, BG11+ YE, BG11 + ACE + YE and BBM. It was found that the fast cell proliferated in the first 2 day’s cultivation in the five medium is resulted from motile cells released from sporangium. After that, the motile cells in BG11 and BG11+ACE medium transform to non-motile cells. In contrast, most cells in BBM,BG11+ YE and BG11+ACE +YE medium are still in motile stage. Such differences in cell propagation and differentiation are contributed by mitosis and the formation of sporangium of motile cells, and the lack of motile cell propagation in BG11 and BG11+ACE medium is produced by the low phosphorus concentration.
Finally, cell fusion was applied to H. pluvialis and Chlorella by PEG-mediated method, and the fusants (Hp-CK) of the two algae that can grow heterotrophicly on sugar were obtained and screened. However, the genetic instability results in the loss of the obtained phenotype for some fusants during the continued division. It also proved the possibility to obtain Haematococcus strain that has the ability of heterotrophic growth on sugar. Furthermore, if the cell fusion could combine with mutagenesis to achieve stable inheritance of genomic DNA, the breeding efficiency of heterotrophic phenotype strain would be further improved.
摘要: 雨生红球藻(Haematococcus pluvialis)被公认为自然界中生产天然虾青素的最好生物,但其生长缓慢,培养条件要求高,细胞无法进行高密度培养,这些都限制了雨生红球藻的规模培养效率。而单细胞绿藻小球藻(Chlorella)环境耐受性强、繁殖快、人工培养较易,而且能够利用葡萄糖等多种底物进行高密度异养发酵。因此,如果能够通过它们之间的细胞融合杂交过程繁育雨生红球藻新品种,使其既能进行高效糖异养,又能高效积累虾青素,则有可能大大提高雨生红球藻的虾青素生产效率。因此本文以实现小球藻和雨生红球藻的细胞融合与糖异养突变株筛选为目标,建立了雨生红球藻原生质体制备技术,分析了雨生红球藻细胞分化的影响以及调控因素,实现了雨生红球藻与小球藻间原生质体的杂交融合与筛选,并对获得的融合子进行了表型鉴定与分析。
首先,通过雨生红球藻的原生质体制备研究发现,雨生红球藻生活史中的游动细胞与不动细胞分化对原生质体的制备过程有明显的影响,因此需要针对性采取不同的酶处理条件。相对于雨生红球藻不动细胞,游动细胞缺少纤维素化的细胞壁结构,原生质体仅由胞外基质(ECM)所包被,这使得游动细胞本身就对外界的低渗透压环境反应敏感。由于细胞在低渗透压下的爆破特点是原生质体制备效率的关键指标,因而培养中高游动细胞比例经常会被误认为获得了高的原生质体制备效率。为此,本研究分别利用富集化培养获得的雨生红球藻游动细胞和不动细胞,对比了不同酶处理过程的影响,建立了相关的原生质体制备与处理技术。结果表明,雨生红球藻游动细胞经过蛋白酶处理后,ECM层被去除,得到完全裸露的原生质体,其中以0.4%的胶原蛋白酶IV效果最佳,在35℃下酶解45 min后,ECM去除率为74.43%,细胞存活率为69.16%;不动细胞的原生质体制备中,以蛋白酶K和纤维素酶+蜗牛酶的组合效果最佳,酶处理后渗透压敏感细胞的得率可以提高到40%左右,但是显微观察证明,虽然不动细胞的细胞壁纤维素层得到有效降解,但是其二级细胞壁结构仍然大部分保持完整,这限制了其原生质体制备效率的进一步提高。
其次,本文基于游动细胞和不动细胞间Triton X-100敏感性差异指标,建立了一种快速监测培养过程中细胞分化的方法,并利用该方法对雨生红球藻在五种培养基BG11、BG11+ACE、BG11 +YE、BG11+ACE +YE和BBM中的生长与细胞分化差异进行了比较分析。发现雨生红球藻在五种培养基中培养的前2天出现的细胞数快速增殖主要是不动细胞形成孢子囊,再释放出游动孢子的繁殖模式。但在培养2天以后,BG11和BG11+ACE培养基中大部分游动孢子都成熟并转变成不动细胞;而BBM、BG11 +YE和BG11+ACE+YE培养基中游动细胞在后续的几天培养中仍维持较高比例。这种细胞分化差异主要是由于BBM、BG11 +YE和BG11+ACE+YE培养基中游动细胞通过有丝分裂或形成游动孢子囊两种模式维持了其继续分裂与增殖的能力;而BG11和BG11+ACE培养基中这些繁殖形式缺失的主要原因在于其较低的磷浓度。因此培养基中适当高的磷含量对于保持雨生红球藻培养中期游动细胞的持续繁殖非常关键。
最后,通过PEG介导的方法进行了雨生红球藻与小球藻原生质体的融合杂交,并且通过选择性的筛选条件,获得了能够进行糖异养生长的雨生红球藻与小球藻融合子(Hp-CK)克隆。通过初步鉴定结果发现部分融合子在持续分裂后存在遗传不稳定的特点,从而导致所获杂交表型的减弱和丧失,但是该结果表明通过雨生红球藻与小球藻的原生质体杂交有可能获得具有利用葡萄糖生产的雨生红球藻异养表型株,而且如果能对原生质体融合株进行进一步的诱变或其他手段介导,实现基因组DNA间的稳定杂交遗传,将有可能进一步提高异养表型株的育种效率。2017-12-29T12:01:15Z