摘要:
以日本沼虾肌肉组织为材料，提取其基因组DNA，研究了该虾16S rRNA基因片段扩增的PCR反应条件.经对扩增反应体系中各因子和扩增程序的梯度实验，确定优化反应条件为: 在25μL反应体系中，10×Buffer 2.5 μL，DNA模板200～300 ng，4×dNTP 0.2 mmol/L，Mg2+1.5 mmol/L，引物0.4μmol/L，Taq DNA聚合酶1 U，95 ℃预变性5 min；接着35个循环包括95 ℃变性1 min，56 ℃复性50 s，72 ℃延伸1 min；最后72 ℃延伸10 min.应用此优化体系扩增日本沼虾18S rRNA，细胞色素氧化酶亚基I (cytochrome oxidase subunit I, COI),NADH脱氢酶亚基V (NADH dehydrogenase subunit 5, ND5)等基因片段均获得了稳定而理想的效果.研究结果为深入探讨日本沼虾种群的遗传和变异,以及开展其种质鉴定和分子标记等研究提供了参考.
Abstract:
Genomic DNA was isolated from the musculature of Macrobrachium nipponensis to establish a reproducible and stable PCR technical system. The ingredients and programs of amplified reactions were optimized respectively. The results showed that the profiles of PCR products could be improved obviously by using the 25 μL solution of 10×Buffer 2.5 μL, Mg+2 1.5 mmol/L, 4×dNTP 0.2 mmol/L, 1 U Taq DNA polymerase, 0.4 μmol/L of each primer and 200~300 ng of DNA template as well as the programs of 5 min at 95 ℃, 35 cycles of 1 min at 95 ℃, 50 s at 56 ℃, 1 min at 72 ℃ and finally, 10 min at 72 ℃. The optimized conditions could result in satisfactory specificity and reproducibility in amplifying 18S rRNA,COI,ND5 gene fragments, and consequently provide a useful means for the research of genetic diversity, molecular marker and germplasm identification in Macrobrachium nipponensis.
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