摘要
以油菜籽、芝麻和亚麻籽为原料,螺旋压榨法制油,采用乳化法、富集法、十六烷基三甲基溴化胺(CTAB)法、树脂试剂盒法和冷冻干燥法提取植物油DNA,并通过超微量可见分光光度计、实时聚合酶链式反应(PCR)、微卫星(SSR)分子标记评价DNA质量.结果表明CTAB法提取的DNA质量浓度高、纯度好、耗时短、起始油量低、费用低、适合分子分析.选择CTAB法,考察起始油量、有机化合物添加、裂解缓冲液组成、裂解和沉淀时间的提取效率.优化条件为最低起始油量100 μL,有机化合物添加CTAB-氯仿:异戊醇(CTAB-C:I),裂解缓冲液组成为CTAB-β巯基乙醇-蛋白酶K(CTAB-βME-PK),裂解20 min,沉淀9 h.该方法在7种精炼植物油中提取到适合分子溯源的DNA,为植物油可追溯性和真实性检测提供了技术支撑.
Abstract
With rapeseed,sesame and flaxseed as the raw materials,the oil was produced by screw pressing,the vegetable oil DNA was extracted by emulsification,enrichment,CTAB,resin kit and freeze-drying methods,and the DNA quality was evaluated by ultra-microscopic visible spectrophotometry,real-time polymerase chain reaction(PCR),and microsatellite(SSR)molecular markers.The results indicated that the CTAB method extracts DNA with high concentration,good purity,short time consuming,low starting oil,low cost and suitable for molecular anal-ysis.The CTAB method was chosen to investigate the extraction efficiency of starting oil volume,organic compound addition,lysis buffer composition,lysis and precipitation time.Optimized conditions were a minimum starting oil vol-ume of 100 μL,organic compound addition:CTAB-chloroform:isoamyl alcohol(CTAB-C:I),lysis buffer com-position:CTAB-β mercaptoethanol-proteinase K(CTAB-βME-PK),lysis for 20 min and precipitation for 9 h.The method has extracted DNA suitable for molecular traceability in seven refined vegetable oils,providing technical support for traceability and authenticity testing of vegetable oils.
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