基于CRISPR/Cas9技术研究金针菇冷诱导结实基因HK1和HK2的编辑转化系统

Establishment of a CRISPR/Cas9 System For Editing Cold-induced Gene HK1/HK2 in Flammulina velutipes

对金针菇(Flammulina velutipes)冷诱导相关的组氨酸激酶基因HK1和HK2构建了基因组编辑(CRISPR/Cas9)pgfvs-Cas9-HK1-gRNA和pgfvs-Cas9-HK2-gRNA表达载体,探索了金针菇菌丝体及原生质体对潮霉素的最低敏感浓度,利用PEG介导的方法将表达载体pgfvs-Cas9-HK1/HK2-gRNA转化金针菇原生质体,经潮霉素筛选后的拟转化子再进行PCR鉴定。结果显示:金针菇菌丝体及原生质体对潮霉素的最低敏感浓度均为50μg/mL;经潮霉素筛选后的部分拟转化子成功扩增出Cas9的部分片段,携带有HK1、HK2基因载体的Cas9蛋白基因成功整合到了金针菇的基因组中,两个表达载体转化率分别为24.1%和12.5%。

A CRISPR/Cas9 system was established for editing two histidine kinase genes HK1 and HK2 that involve in fruiting body development in Flammulina velutipes.Plasmids harboring cas9 and target gene HK1-gRNA and HK2-gRNA were constructed to generate pgfvs-Cas9-HK1-gRNA and pgfvs-Cas9-HK2-gRNA,respectively.Then the plasmids were transformed into F.velutipes by PEG mediated protoplast transformation.Regenerated colonies were first screened by hygromycin and then screened by PCR to confirm the existence of cas 9 gene in transformants.The results showed that the threshold concentration of hygromycin effective to F.velutipes protoplast was 50μg/mL.The cas9 gene was detected in positive transformants for both HK1 and HK2 plasmids,suggesting both plasmids were successfully transformed into F.velutipes.Transformation rates of pgfvs-Cas9-HK1-gRNA and pgfvs-Cas9-HK2-gRNA were 24.1%and 12.5%,respectively.