转基因玉米植株幼叶DNA快速提取及降落PCR检测研究

用农杆菌介导玉米自交系‘掖478’茎尖遗传转化的转基因玉米植株幼叶为材料,进行快速提取玉米叶片DNA方法的研究.在室温下取2cm2大小的玉米叶片,将叶片剪碎置于2.0mL离心管中,液氮处理后使用组织研磨器研磨,然后加入800μL CTAB提取液,24∶1的氯仿∶异戊醇溶液800μL,轻摇10min后于10 000r/min离心5min,取上清,最后用0.7倍体积冰异丙醇沉淀DNA.应用降落PCR进行目的基因片段的扩增,并对结果进行测序验证.结果表明,该方法操作简单,省时省力,节约成本,效率高,降落PCR扩增目的基因条带清晰,测序结果与原序列一致.

Rapid extraction and touchdown PCR detection of DNA from seedling leaves of transgenic maize

Leaves of 24 transgenic seedlings of maize inbred line ’Ye478’ transformed by Agrobacterium tumefaciens were taken as the experimental materials,a rapid extraction of DNA from maize leaves was constructed.To do this,2 cm2 size leaf of maize was collected into 2.0 mL centrifugation tube through high-throughput tissue grinder under normal temperature,and grinded the leaves after liquid nitrogen treated,then adding 800 μL CTAB extraction and 800 μL chloroform∶isoamyl alcohol(24∶1) into the tube.The clear liquid was taken up after centrifugated with 10 000 r/min for 10 min.Finally,DNA was precipitated by freezing isopropanol,and the target gene fragments were amplified by the touchdown PCR and sequenced.The results showed that this method was simple,would save time and cost,and was high efficiency.Bands of target gene amplified by touchdown PCR were clear,and the sequencing results were compared to verify its correctness.