【作者】 尉玮； 王丽华；

【Author】 WEI Wei;WANG Lihua;Bee Science School,Fujian Agriculture and Forestry University;

【机构】 福建农林大学蜂学学院；

【摘要】 本研究选取了几个重要的胚胎发育基因,分别构建了TALEN和CRISPR/Cas9打靶载体。采用单元组合法对En、Prd和Tll合成了TALEN靶位点的左臂和右臂,并体外转录合成了各自TALEN的mRNA:采用体外转录法对Dfd、Eve、Otd-1、Prd和Tll制备出含有CRISPR/Cas9靶位点的gRNA模板,并回收得到各自的Cas9/gRNA。利用显微注射技术将目标基因-TALEN mRNA或目标基因-Cas9/gRNA分别导入到意大利蜜蜂5-6h的卵中,在35℃下培养48-60h后,进行限制性内切酶酶切检测。结果显示:对目标基因-TALEN或目标基因-Cas9/gRNA扩增片段TA克隆测序后,发现Tll-TALEN和Otd-1-Cas9/gRNA都存在着单碱基突变,表明TALEN和CRISPR/Cas9编辑技术有望用在蜜蜂基因组中进行基因靶向诱变。更多还原

【Abstract】 Several important embryonic development genes were selected to construct into TALEN-or CRISPR/Cas9-targeting vectors.The TALEN mRNA of left or/and right arms for the En-,Prd-or Tll-TALEN targeting point was synthesized by unit assembly method and then transcripted in vitro.The gRNA template containing CRISPR/Cas9 targeting point for the Dfd,Eve,Otd-1,Prd or Tll was also prepared by transcription in vitro and then the Cas9/gRNA was collected.Target gene-TALEN mRNA or target gene-Cas9/gRNA was microinjected into honeybee(Apis mellifera)embryos at 5-6 h,the gDNA was assayed by restriction enzyme after the embryos were incubated at 35 ℃ for 48-60 h.The results showed that a single-base mutation was found on Tll-TALEN or on Otd-1-Cas9/gRNA by sequencing TA cloning of TALEN-or Cas9/gRNA-PCR fragment,suggesting TALEN or CRISPR/Cas9 editing be hopeful to induce gene-targeted mutation in honeybee genome.更多还原