利用CRISPR/Cas9技术靶向编辑青花菜BoZDS

Targeted Editing of BoZDS in Broccoli by CRISPR/Cas9 Technology

胡萝卜素脱氢酶(ζ-carotene desaturase,ZDS)是类胡萝卜素合成的限速酶。本试验以青花菜多代自交系‘ZN09’为试材,以BoZDS为目标基因,在其第1个外显子上选取2个靶位点,分别构建CRISPR/Cas9载体进行稳定遗传转化。1号靶位点转化效率为0.80%,突变率为15.79%,共获得3株突变体,均为杂合突变;2号靶位点转化效率为0.84%,突变率为36.84%,共获得7株突变体,其中纯合突变2株,杂合突变2株,嵌合突变3株。突变体均出现白化或斑驳表型,突变体L*值和a*值均显著高于野生型植株,b*值均下降。本试验建立了青花菜CRISPR/Cas9基因编辑稳定遗传体系,并对BoZDS基因进行有效编辑,研究结果为利用基因编辑技术进行青花菜基因功能研究与优异性状材料创制提供了技术支撑。

Carotene desaturase(ZDS)is one of the key enzymes regulating carotenoids biosynthesis.Here having the high-generation inbred broccoli(Brassica oleracea var.italica)line’ZN09’as testing materials and BoZDS as target gene,then two target sites on the first exon of BoZDS were selected to construct CRISPR/Cas9 vector for stable genetic transformation.For the target site 1,the transformation efficiency and the mutation rate were 0.80%and 15.79%,respectively,and three heterozygous mutants were generated.For the target site 2,the transformation efficiency was 0.84%,and the mutation rate was 36.84%,and seven mutants were generated,including two homozygous,two heterozygous,and three chimeric mutants.All of the mutant plants showed albino or mottled phenotype.The L*and a*values of mutant plants were significantly higher than those of wild-type plants,while the b*value decreased.To sum up,a stable genetic system for CRISPR/Cas9-mediated gene editing in broccoli was established,and BoZDS was effectively edited.Our results provide the technical support for gene function study and germplasm innovation of broccoli by using gene editing technology.