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
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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%。
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.
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.
引文
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[15]刘建雨,徐珍,张丹,等.农杆菌介导法转化金针菇不同受体的效率比较[J].食用菌学报,2015,22(3):7-12.
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[2]王泽华.金针菇食用价值初探[J].农产品加工,2015(5):47-48.
[3]刘景煜.金针菇生物活性成分分离及开发利用[D].杨凌:西北农林科技大学,2017.
[4]庞杰,孙国琴,于静,等.35种食用菌菌丝生长和子实体分化温度的聚类分析[J].中国食用菌,2015,34(6):34-37.
[5]SAKAMOTO Y,TAMAI Y,YAJIMA T.Influence of light on the morphological changes that take place during the development of the Flammulina velutipes fruit body[J].Mycoscience,2004,45(5):333-339.
[6]张江萍.杏鲍菇工厂化栽培存在的问题及对策[J].现代农业科技,2012(1):156-157.
[7]MEJEAN V.Two-component regulatory systems:The moment of truth[J].Research in Microbiology,2016,167(1):1-3.
[8]曹云飞,张海娜,肖凯.CBF转录因子介导的植物低温信号转导研究进展[J].棉花学报,2007,19(4):304-311.
[9]金华燕.蛹虫草组氨酸激酶基因(CmHK)和Kexin基因(CmKexin)的研究[D].南昌:江西师范大学,2013.
[10]LATORRE A,SOMOZA.Modified RNAs in CRISPR/Cas9:An old trick works again[J].Angewandte Chemie International Edition,2016,55(11):3548-3550.
[11]RAN FA,HSU PD,LIN C,et al.Double Nicking by RNA-Guided CRISPR Cas9for enhanced genome editing specificity(vol 154,pg 1380,2013)[J].Cell,2013,155(02):479-480.
[12]RAN FA,HSU PD,WRIGHT J,et al.Genome engineering using the CRISPR-Cas9system[J].Nature Protocols,2013,8(11):2281-2308.
[13]景润春,卢洪.CRISPR/Cas9基因组定向编辑技术的发展与在作物遗传育种中的应用[J].中国农业科学,2016,49(7):1219-1229.
[14]龚淑俐,邓放明,陈力力.金针菇原生质体制备的研究[J].现代食品科技,2007,23(4):54-57.
[15]刘建雨,徐珍,张丹,等.农杆菌介导法转化金针菇不同受体的效率比较[J].食用菌学报,2015,22(3):7-12.
[16]DICARLO JE,NORVILLE JE,MALI P,et al.Genome engineering in Saccharomyces cerevisiae using CRISPRCas systems[J].Nucleic Acids Research,2013,41(7):4336-4343.