螺旋藻(Spirulina)基因组外DNA的高效提取与纯化

HIGH EFFICIENT EXTRA-GENOMIC DNA EXTRACTION AND PURIFICATION FROM SPIRULINA

通过研究建立了一种简便、高效提取螺旋藻基因组外DNA(exDNA)的方法——CTAB-蛋白酶K法。该法先以含200μg/ml蛋白酶K的CTAB提取液裂解螺旋藻细胞,并用氯仿/异戊醇抽提得到总DNA粗提物,粗提物再经100μg/ml蛋白酶K酶解及酚/氯仿/异戊醇抽提得到总DNA,再利用凝胶冻融离心法得到高纯度exDNA。利用上述方法,对6株钝顶螺旋藻品系Sp-1、Sp-2、Sp-3、Sp-4、Sp-5和Sp-6所作的研究结果表明,(1)6株品系均含有exDNA,但数目与相对质量不尽相同;(2)Sp-1、Sp-2、Sp-5和Sp-6均只有一种exDNA,依次约为1.15kb、0.75kb、1.15kb和1.1kb;(3)Sp-3和Sp-4均有2条exDNA带,Sp-3的exDNA约为1.55kb和3.0kb,Sp-4的exDNA约为1.8kb和3.6kb;(4)螺旋藻品种(系)间exDNA的异同性及其与生理生态等特性的关系,暗示着exDNA可能与螺旋藻的分类、进化和形态建成等重大生物学课题有关,并有可能担负着某些生物学功能。

Genus Spirulina is a photoautotrophic filamentous cyanobacterium of Family Oscillatoriaceae, Phylum Cyanophyta. Because of Spirulina contain bioactive polysaccharides, β-carotene and γ-linolenic acid, having superior physiological effects on human, FAO of the UN and WHO claimed it as an ideal food and dietary supplement in the 21st century. Happily, Spirulina is easy to culture in large scale. Because of its alkaliphilic nature, it can be grown as mono-culture in natural environment. Two widely exploited economic microalgae, Spirulina platensis and Spirulina maxima, have been selected for mass production. To optimize the industrial cultivation, genetic study on the microalgae is necessary, and some technologies have been developed in several laboratories. However, conventional approaches such as chromosomal integration and conjugal transfer were not fully successful, because the multi-genome system in Spirulina hampers high expression of exogenous genes, and no endogenous extra-genomic DNA （exDNA） that is suitable to be used as recombinant vector has been found in Spirulina genus. It is not easy to extract and purify exDNA from Spirulina due to its high contents of protein and polysaccharide. In this study, a modified CTAB-Proteinase K extracting method for exDNA in Spirulina is established. First, the Spirulina cells are lysed in CTAB extraction buffer containing 200 μg/ml Proteinase K, and the total DNA crude is extracted with chloroform-isoamyl alcohol. Secondly, the total DNA is obtained after the crude is treated with 100μg/ml Proteinase K and extracted with phenol-chloroform-isoamyl alcohol. Finally, the high-purification exDNA is obtained using gel freezing-thawing method. Furthermore, the exDNA of six strains of Spirulina platensis, Sp-1, Sp-2, Sp-3, Sp-4, Sp-5 and Sp-6, were extracted and analysed. The results show that： （1） all of the six strains contain exDNA, but the number and molecular weight of exDNA are usually different with strains; （2） four stains, Sp-1, Sp-2, Sp-5 and Sp-6, contain only one kind of exDNA, and the molecular weights is about 1.15kb, 0.75kb, 1.15kb and 1.1kb, respectively; （3） Sp-3 and Sp-4 contain two kinds of exDNA, and the molecular weights are about 1.55kb and 3.0kb in Sp-3, and 1.8kb and 3.6kb in Sp-4; （4） the distinction of exDNA in different spirulina strains and its related to physiological and ecological characteristics imply that exDNA may have some biological functions, and relate to taxonomy, evolution, and morphogenesis.