转基因检测中大豆蛋白DNA提取方法筛选及其lectin基因降解分析  

Comparative Evaluation of DNA Extraction Methods for Detection of Transgenic Soybean Protein and Analysis of lectin Gene Degradation during Soybean Protein Extraction

作　　者：杜艳 陈复生[1] 陈晨[1] 刘营 DU Yan;CHEN Fusheng;CHEN Chen;LIU Ying(College of Food Science and Technology,Henan University of Technology,Zhengzhou 450001,China)

机构地区：[1]河南工业大学粮油食品学院

出　　处：《食品科学》2020年第4期102-111,共10页Food Science

基　　金：国家自然科学基金面上项目(21676073;21376064);“十三五”国家重点研发计划重点专项(2018YFD0401100)

摘　　要：采用紫外分光光度法、琼脂糖凝胶电泳法和实时荧光聚合酶链式反应(polymerase chain reaction,PCR)等检测DNA浓度、纯度、完整性和反应灵敏度,综合评价Nucleo试剂盒、Wizard试剂盒、Dneasy试剂盒、Ezup试剂盒、Dzup试剂盒和十二烷基硫酸钠(sodium dodecyl sulfate,SDS)法对大豆蛋白及大豆籽粒中DNA的提取效果,旨在筛选出适用于大豆分离蛋白(soybean protein isolate,SPI)、大豆浓缩蛋白(soybean protein concentrate,SPC)及大豆籽粒中DNA提取的最佳方法,提高转基因大豆检测的精确性和准确性。结果表明,Nucleo试剂盒和Dneasy试剂盒能够得到纯度高、完整性好的DNA,符合实时荧光PCR的检测要求。Dneasy试剂盒提取的DNA纯度更高,更有利于提高后续PCR的重复性、稳定性和精确性,普遍适用于SPI、SPC以及大豆籽粒中DNA的提取。Wizard试剂盒、Ezup试剂盒和SDS法提取效果适中,SDS法成本最低,但耗时最长。Dzup试剂盒得到的DNA虽然浓度高,但含杂质较多,对实时荧光PCR具有抑制作用,且无法保持完整的基因组DNA,不适用于大豆转基因检测中的DNA提取。采用Dneasy试剂盒提取得到的SPI和SPC样品DNA,对不同长度lectin基因扩增结果不同,表明在蛋白制备过程中lectin基因可能存在降解。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.

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 

分 类 号：TS201.6[轻工技术与工程—食品科学]