关键词: 大豆分离蛋白, 大豆浓缩蛋白, 大豆籽粒, DNA提取, 实时荧光聚合酶链式反应

Abstract: In this paper, six methods of DNA extraction, namely Macherey-Nagel Nucleo Spin Kit (Nucleo kit), Wizard Genomic DNA Purification Kit (Wizard kit), Dneasy Plant Mini Kit (Dneasy kit), Ezup column plant genomic DNA extraction kit (Ezup kit), Dzup genomic DNA extraction kit (Dzup kit) and sodium dodecyl sulfate (SDS) method, were comparatively applied to extract DNA from soybean protein extracts and soybean seeds. The DNA concentration, purity and integrity were determined by ultraviolet spectrophotometry, agarose gel electrophoresis and real-time fluorescent PCR, and the reaction sensitivity was evaluated. We aimed to find the optimum method to extract DNA from soybean protein isolate (SPI), soybean protein concentrate (SPC) and soybean seeds so as to improve the sensitivity and accuracy of transgenic soybean detection. Results showed that the purity and integrity of DNA extracted with Nucleo kit and Dneasy kit were high enough for real-time PCR detection. The purity of DNA obtained by Dneasy kit was the highest, being beneficial for improving the repeatability, stability and accuracy of subsequent PCR reaction, so that Dneasy kit was applicable to SPI, SPC and soybean seeds. The extraction efficiencies of Ezup kit and SDS method were moderate. SDS method costed the least but took the longest time. Despite the high yield, the DNA obtained with Dzup kit was fragmentary and contained lots of impurities, which could limit the applicability of Dzup kit for DNA extraction for transgenic soybean detection. Furthermore, the lectin gene was inferred to be degraded during SPI and SPC preparation with Dneasy kit.

Key words: soybean protein isolate, soybean protein concentrate, soybean seeds, DNA extraction, real-time PCR