【摘要】 本研究通过Cre-loxP系统,构建了Kan基因的删除载体系统,利用花青素合成途径中转录因子的表达指示删除的效果。首先,利用两次PCR方法对pGreen载体进行改造,在卡那霉素抗性(Kan)基因的两侧加入两个同向的loxP位点,在多克隆位点前插入5-烯醇式丙酮酸莽草酸-3-磷酸(EPSP)合酶的表达框作为筛选标记基因,命名为pBAC823。在此载体基础上,构建了两个植物表达载体,组成一个抗生素可删除的载体系统。其一是在pBAC823载体Kan基因的一侧加入花青素合成途径中两个转录因子bi基因和cl基因的表达框,命名为pBAC9008。该载体可以作为基础植物表达载体,用于表达目的基因。其二是含Cre酶基因表达框的植物表达载体。将hsp70启动子驱动的cre基因的表达框插入pBAC823载体Kan基因的一侧,并将玉米花青素基因合成途径中转录因子bi和cl的表达框插入多克隆位点,命名为pBAC9009,此载体作为一个删除载体,单独转化植物,之后可以通过杂交方法删除目的基因表达载体中两个loxP位点之间的Kan基因片段。将上述载体转化玉米幼胚,得到T0代转基因玉米的种子,其中部分籽粒为紫色,分子检测结果表明紫色籽粒有花青素基因的插入,验证了花青素合成基因作为可视化标记的可靠性。本研究采用了可视化标记跟踪外源基因的表达和抗生素抗性基因的删除,不仅大大降低了外源基因的检测成本,为转基因食品安全提供技术保证,在转基因研究中具有重要的意义。更多还原

【Abstract】 Based on Cre-loxP recombination system,an antibiotic resistance genes deletion system(Kan) was constructed.To observe the transgene and deletion efficiency,the transcription factor bi and cl genes participated in the anthocyanins synthesis pathway was used as a visible marker in this study.By using polymerase chain reaction two times,the binary vector of pGreen was modified by adding the loxP sequences to each side of kanamycin resistance gene with the same direction.Then,the CaMV 35S promoter drived 5-Enolpyruvylshi-kimate-3-phosphate syntheses(EPSP) gene,a marker gene to select transformed plant cells,was inserted to the NdeⅠ site of the modified pGreen.The latter vector,named pBAC823,was used as a basis to construct the deletion system,which contains the following two vectors.The first one,named pBAC9008,was constructed by inserting maize bi and cl transcription factor,which participated in the anthocyanins synthesis,to the side of pBAC823.The carrier can be used as a basic plant expression vector,for the expression of target genes.The second one,named pBAC9009,which was used to express cre(causes recombinase) gene and delete the kanamycin resistance gene,was constructed by inserting both the hsp70 promotor drived cre gene to the side of pBAC823 and bi and cl box to the multiple cloning sites.It is used as a deleted vector to transform plant alone.Then,target gene expression vector between two loxP sites Kan gene fragment was deleted by hybrid method.The above vectors were used to transform immature maize embryo to obtain transformed T0 maize seed.Part of them were purple.Molecular detection showed that the novel bi and cl genes were integrated into the maize genome of purple seeds.It verified anthocyanin synthesis genes could be used as a reliable visual marker.By adopting the visualization marker tracking exogenous gene expression and antibiotic resistance gene deletion,this study not only greatly reduces the exogenous gene testing cost,but also provides technology guarantee for genetically modified food safety,and has a significant meaning in transgenic study.更多还原