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

欧阳萍兰, 李琼洁, 郭丽琼, 林俊芳, 叶志伟, 魏韬, 郑倩望, 伍土恒, 罗润

食用菌学报. 2018, 25(03): 1-7

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食用菌学报 ›› 2018, Vol. 25 ›› Issue (03) : 1-7. DOI: 10.16488/j.cnki.1005-9873.2018.03.001
研究论文

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

  • 欧阳萍兰, 李琼洁, 郭丽琼, 林俊芳, 叶志伟, 魏韬, 郑倩望, 伍土恒, 罗润
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Establishment of a CRISPR/Cas9 System For Editing Cold-induced Gene HK1/HK2 in Flammulina velutipes

  • OUYANG Pinglan, LI Qiongjie, GUO Liqiong, LIN Junfang, YE Zhiwei, WEI Tao, ZHENG Qianwang, WU Tuheng, LUO Run
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摘要

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%。

Abstract

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% and12.5%,respectively.?

关键词

金针菇 / CRISPR/Cas9 / PEG介导的转化

Key words

Flammulina velutipes / CRISPR/Cas9 / PEG mediated transformation

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欧阳萍兰, 李琼洁, 郭丽琼, 林俊芳, 叶志伟, 魏韬, 郑倩望, 伍土恒, 罗润. 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. 食用菌学报. 2018, 25(03): 1-7 https://doi.org/10.16488/j.cnki.1005-9873.2018.03.001
OUYANG Pinglan, LI Qiongjie, GUO Liqiong, LIN Junfang, YE Zhiwei, WEI Tao, ZHENG Qianwang, WU Tuheng, LUO Run. Establishment of a CRISPR/Cas9 System For Editing Cold-induced Gene HK1/HK2 in Flammulina velutipes. Acta Edulis Fungi. 2018, 25(03): 1-7 https://doi.org/10.16488/j.cnki.1005-9873.2018.03.001
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