菠萝U6启动子克隆及活性分析

Cloning and Activity Analysis of U6 Promoter in Pineapple

单链向导RNA(single guide RNA,sgRNA)是CRISPR/Cas9系统的“指挥中心”,可以引导Cas9对DNA进行定点编辑,其转录能力直接影响基因编辑效率。U6启动子常用于驱动sg RNA的表达,尤其是本物种内源U6启动子通常具有更高效的启动效率。为了筛选出菠萝内源高效转录活性U6启动子,提升菠萝CRISPR/Cas9基因编辑效率,本研究从菠萝基因组中克隆了5个内源U6启动子,选择水稻3个U6启动子,构建驱动萤火虫荧光素酶基因(firefly luciferase,LUC)的融合表达载体,瞬时转化本氏烟草叶片,通过检测生物化学发光活性,比较各个启动子的转录活性。研究结果表明:从菠萝基因组克隆出5个U6启动子分别命名为:AcU6-1P、AcU6-2P、AcU6-3P、AcU6-4P、AcU6-5P;生物化学发光信号检测发现5个AcU6均具有转录活性,其中AcU6-2P启动子驱动LUC表达的荧光信号最为强烈,其次为AcU6-5P启动子;生物化学发光活性分析发现AcU6-2P启动子驱动产生的荧光素酶活性最高,AcU6-5P仅次于AcU6-2P,结果与荧光信号一致。综上,从菠萝基因组中筛选出2个具有高效转录活性的内源U6启动子AcU6-2P和AcU6-5P,可为后续菠萝CRISPR/Cas9基因编辑系统的优化和分子育种提供了依据。

Single guide RNA(sgRNA)is the"command center"of CRISPR/Cas9 system,which can guide Cas9 to edit DNA at a fixed point.Its transcription ability directly affects the efficiency of gene editing.U6 promoters are often used to drive the expression of sgRNA,especially the endogenous U6 promoters of own species usually have more efficient starting efficiency.In order to screen out the endogenous high-efficient transcriptional activity U6 promoter of pineapple and improve the gene editing efficiency of pineapple CRISPR/Cas9,five endogenous U6 promoters were cloned from the pineapple genome,three U6 promoters were selected from rice,and a fusion expression vector driving firefly luciferase(LUC)gene was constructed,the tobacco leaves was transiently transformed,and the transcriptional activity of each promoter was compared by detecting the biochemical luminescence activity.The results showed that five U6 promoters were cloned from pineapple genome and named AcU6-1P,AcU6-2P,AcU6-3P,AcU6-4P and AcU6-5P respectively;Biochemical luminescence signal detection found that five AcU6 have transcriptional activity,of which the AcU6-2P promoter drives the strongest fluorescence signal of LUC expression,followed by AcU6-5P promoter;Biochemical luminescence activity analysis showed that the activity of luciferase driven by AcU6-2P promoter was the highest,and AcU6-5P was second only to AcU6-2P,which was consistent with the fluorescence signal.To sum up,two endogenous U6 promoters AcU6-2P and AcU6-5P with high transcriptional activity were screened from the pineapple genome,which provided a basis for the optimization of the subsequent pineapple CRISPR/Cas9 gene editing system and molecular breeding.