应用单细胞rDNA序列测定方法鉴定不同生活史阶段的亚历山大藻

Application of single-cell rDNA sequence analysis in identification of Alexandrium cells at various life stages

通过实验室内诱导产生了重要的赤潮原因种链状亚历山大藻(Alexandrium catenella)不同生活史阶段的藻细胞,建立了低渗(TE缓冲液)溶胀和蛋白酶K处理相结合的细胞裂解方法和单细胞PCR扩增方法。应用所建立的方法,测定了不同生活史阶段的亚历山大藻细胞核糖体DNA(rDNA)两个高变区域(5.8S rDNA及其两侧的内转录间隔区和28S rDNA 5’端D1-D2区)的序列信息。结果显示,各生活史阶段的藻细胞rDNA序列在所测的两个高变区域完全相同,依据序列信息可以对处于不同生活史阶段的亚历山大藻进行鉴定。同时发现,在单个藻细胞中,两个所测片段均存有若干个多态位点,表明藻细胞内rDNA序列的多个拷贝之间可能存有差异。实验表明,对处于不同生活史阶段的赤潮原因种,通过单细胞rDNA序列测定的方法也可以进行准确鉴定。

Alexandrium catenella, an important HA_B-causative species, was used to test the feasibility of a single-cell ribosomal DNA （rDNA） sequence analytical method in identification of target algal cells, particularly at various life stages. Alexandrium cells at various life stages were induced and examined in the laboratory and the DNA template for PCR am- plification was prepared from a single cell of A. catenella using the combination of low-osmotic TE buffer and proteinase K treatment. Two high diverse regions of nuclear-encoded rDNA, the 5.8S rDNA and its flanking imernal transcribed spacers 1 and 2, and the 5＇ portion of the large-subunit rDNA were then amplified and sequenced. It was found that the rDNA sequences of Alexandrium cells at different life stages were all the same in the two high diverse regions. Therefore, Alexandrium cells at various life stages could be idemified based on the sequence analysis of rDNA. Besides, polymorphism was found existing in several sites of rDNA sequences from a single cell.