雨生红球藻原生质体制备及细胞融合研究

QIBEBT-IR > 微藻生物技术研究组

	雨生红球藻原生质体制备及细胞融合研究
其他题名	无
	徐晓莹
导师	刘天中
	2015-06
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	生物化工
关键词	雨生红球藻原生质体制备繁殖方式小球藻细胞融合
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。
其他摘要	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.
作者部门	微藻生物技术团队
学科领域	工学
公开日期	2018-09
学位类型	硕士 ; 硕士学位论文
语种	中文
文献类型	学位论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/9783
专题	微藻生物技术研究组
作者单位	1.中国科学院 2.青岛生物能源与过程研究所
推荐引用方式 GB/T 7714	徐晓莹. 雨生红球藻原生质体制备及细胞融合研究[D]. 北京. 中国科学院大学,2015.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
雨生红球藻原生质体制备及细胞融合研究.p（2649KB）	学位论文		开放获取	CC BY-NC-SA	请求全文