以赤潮种裸甲藻(Gymnodiniumsp.)和微小原甲藻(Prorocentrumminimum)为试验材料,以不同方法提取 其基因组并进行纯化,然后采用PCR方法扩增其rRNA基因,包括18S,28S和ITS片断,并进行扩增条件的比较和优化,得到两种藻的最佳DNA提取条件和PCR扩增条件.裸甲藻和微小原甲藻的DNA提取宜采用改良的CTAB 方法;并需对粗提取的DNA用CTAB方法进行纯化.两种藻的最适模板浓度为纯化后模板1 0～2.0μL;最适Mg2+ 浓度为2 0μL(25mmol/L);ITS引物PCR扩增的退火温度为50℃,而18S三对引物的退火温度均为55℃,28S的退 火温度为54℃最为适宜.The genomes from bloom-forming species, Gymnodinium sp.and Prorocentrum minimum were extracted and purified by a series of methods, and the gene fragments of 18S,28S and ITS (Internal Transcribed Spacer) of Gymnodinium sp.and Prorocentrum minimum genomic DNA were amplified by polymerase chain reaction(PCR).All of the extracting methods and PCR condition were optimized by comparing its advantages, and the preferable methodological conditions were achieved. The advisable method of extracting and purifying genome was the improved method of CTAB, and the genomes should be purified by CTAB also.The better concentration of DNA template was 1.0～2.0μL of purified genomes from Gymnodinium sp.and Prorocentrum minimum, and the concentration of Mg~(2+) was 2.0μL of 25 mmol/L in 25μL PCR systems.The advisable annealing temperature was 50℃ to primer pair of ITS, 55℃ to 18S, and 54℃ to 28S respectively in PCR program.国家重点基础研究发展规划973项目(G1999043706);; 国家自然科学基金(40076031;40376032)资助项目
