小球藻细胞壁缺陷型突变体的筛选及转化系统的建立

由于高效、稳定遗传转化系统的缺乏,小球藻遗传改良以及油脂代谢机理的研究等工作难以进行。研究旨在通过筛选获得小球藻Chlorella vulgaris细胞壁缺陷型突变体,在此基础上建立其转化系统。首先通过紫外诱变获得小球藻Chlorella vulgaris的突变体库,基于细胞壁缺陷型突变体在1% Triton X-100处理后叶绿素会释放到上清中的原理,利用酶标仪高通量地从约4000个突变体中筛选获得10株细胞壁缺陷的小球藻。同时以小球藻内源性-tubulin的启动子和终止子作为启动子和终止子,以AphⅧ (Aminoglycoside 3'-Phosphotransferase type Ⅷ)作为报告基因构建了转化载体pHK203。通过优化电转缓冲液组分和电击参数,确定了细胞壁缺陷型突变体CWD-3的最佳转化条件,即2 g pHK203,ddH2O作为电转缓冲液,1500 V,525 ,50 F的电击条件下,转化效率可达到40个转化子/g DNA。研究为小球藻Chlorella vulgaris的油脂代谢通路和遗传改良提供了技术基础,同时由于可降低破壁成本,筛选获得的细胞壁缺陷型突变体适于工业化生产小球藻藻粉。

SCREENING OF CELL WALL-DEFECTIVE MUTANTS AND CONSTRUCTION OF TRANSFORMATION SYSTEM IN CHLORELLA VULGARIS

Chlorella vulgaris has been widely recognized as a safe food ingredient due to its richness in proteins, vita-mins, minerals and short chain poly-unsaturated fatty acid. Recently, Chlorella vulgaris was chosen as one of the feedstock for biodiesel because of its high growth rate and high lipid content, but lacking of stable and efficient transformation system has seriously hindered the understanding of lipid metabolism and the development of the genetic engineering. Our aim was to obtain cell wall-defective mutants of Chlorella vulgaris and use them to construct an electro-transformation system. Based on the release ability of chlorophyll after treatment of 1% Triton X-100, we set up a high throughput screening method of cell wall-defective mutants and obtained 10 cell wall-defective mutants. In addition, we constructed a transformation vector pHK203 by using endogenous promoter and 3'-UTR of -tubulin gene and by using AphⅧ as selective marker. We successfully obtained 40 transformants per g DNA using plasmid pHK203 and ddH2O as electroporation buffer with the best electroporation condition of 1500 V, 525 , 50 F. Our study will benefit the study of lipid metabolism pathway and genetic engineering of Chlorella vulgaris.