金针菇G蛋白偶联受体基因的CRISPR/Cas9基因组编辑载体构建及转化研究
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摘要
通过氨基酸同源比对(Blast P)以及金针菇冷诱导前后菌丝阶段和原基阶段的转录组数据分析,获得了金针菇中的两个假定G蛋白偶联受体基因Fvgpcr1、Fvgpcr2。对获得的金针菇假定G蛋白偶联受体基因Fvgpcr1、 Fvgpcr2构建了基因组编辑(CRISPR/Cas9)的pCAMBIA0390-hph-Fvcas9-Fvgpcr1-sgRNA1/sgRNA2、pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2等4个表达载体。通过农杆菌介导(ATMT)将表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA转化金针菇菌丝体,采用潮霉素和头孢毒素低浓度初筛和高浓度复筛,经两段筛选获得金针菇拟转化子。经对拟转化子进行PCR鉴定、RT-qPCR检测和Western杂交验证,结果显示表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA成功整合进金针菇基因组中,FvCas9蛋白正常表达,但未得到Fvgpcr基因敲除突变体。本研究利用农杆菌介导转化法在金针菇中构建了CRISPR/Cas9敲除体系,对后续目标基因的敲除有着重要意义。
Two putative G protein-coupled receptor genes Fvgpcr1 and Fvgpcr2 in Flammulina filiformis were obtained by amino acid homology alignment(Blast P) and transcriptome data analysis of the fungus in hyphae and primordium stages before and after cold induction. Four expression vectors including p CAMBIA0390-hph-Fvcas9-Fvgpcr1-sg RNA1/sg RNA2 and pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2 were constructed for gene disruption of G-protein coupled receptor gene Fvgpcr1 and Fvgpcr2. The expression vector p CAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA was transformed into the mycelium of F. filiformis by Agrobacterium tumefaciensmediated(ATMT). Transformants harvested from hygromycin and cefotaxime rescreening were identified by PCR, RT-qPCR and Western hybridization. Results showed that the expression vectors pCAMBIA0390-hphFvcas9-Fvgpcr-sgRNA1 and pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA2 were successfully integrated into the genome of F. filiformis and the Fv Cas9 protein was successfully expressed. However, the Fvgpcr deletion mutant was not obtained. This study used the Agrobacterium-mediated transformation method to construct a CRISPR/Cas9 gene disruption system in F. filiformis, which is of great significance for the subsequent gene disruption.
Two putative G protein-coupled receptor genes Fvgpcr1 and Fvgpcr2 in Flammulina filiformis were obtained by amino acid homology alignment(Blast P) and transcriptome data analysis of the fungus in hyphae and primordium stages before and after cold induction. Four expression vectors including p CAMBIA0390-hph-Fvcas9-Fvgpcr1-sg RNA1/sg RNA2 and pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2 were constructed for gene disruption of G-protein coupled receptor gene Fvgpcr1 and Fvgpcr2. The expression vector p CAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA was transformed into the mycelium of F. filiformis by Agrobacterium tumefaciensmediated(ATMT). Transformants harvested from hygromycin and cefotaxime rescreening were identified by PCR, RT-qPCR and Western hybridization. Results showed that the expression vectors pCAMBIA0390-hphFvcas9-Fvgpcr-sgRNA1 and pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA2 were successfully integrated into the genome of F. filiformis and the Fv Cas9 protein was successfully expressed. However, the Fvgpcr deletion mutant was not obtained. This study used the Agrobacterium-mediated transformation method to construct a CRISPR/Cas9 gene disruption system in F. filiformis, which is of great significance for the subsequent gene disruption.
引文
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Cogoni C,Romano N,Macino G,1994.Suppression of gene expression by homologous transgenes.Antonie van Leeuwenhoek,65(3):205-209
Fu Y,Sander JD,Reyon D,Cascio VM,Joung JK,2014.Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.Nature Biotechnology,32(3):279-284
Ge ZW,Liu XB,Zhao K,Yang ZL,2015.Species diversity of Flammulina in China:new varieties and a new record.Mycosystema,34(4):589-603
Hou B,Zheng SY,Tai LM,Sang L,Wu SR,2013.Research on nutrition health function and food processing of Flammulina velutipes.Food Research and Development,34(12):122-126(in Chinese)
Han F,Kang LZ,Zeng XL,Ye ZW,Guo LQ,Lin JF,2014.Bioproduction of baccatin III,an advanced precursor of paclitaxol,with transgenic Flammulina velutipes expressing the 10-deacetylbaccatin III-10-O-acetyl transferase gene.Journal of the Science of Food and Agriculture,94(12):2376-2383
Katayama T,Tanaka Y,Okabe T,Nakamura H,Fujii W,Kitamoto K,Maruyama J,2016.Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae.Biotechnology Letters,38(4):637-642
Li L,Wright SJ,Krystofova S,Park G,Borkovich KA,2007.Heterotrimeric G protein signaling in filamentous fungi.Annual Review of Microbiology,61:423-452
Lu WZ,Wu BL,Zhao Q,2012.G protein coupled receptors,the magic molecules with seven transmembrane helices:a brief introduction to the nobel prize in chemistry 2012.Chinese Journal of Nature,34(6):337-343(in Chinese)
Luo R,Lin JF,Guo LQ,Ye ZW,Guo TF,Yun F,2016.Construction of Flammulina velutipes genome editing vector by using CRISPR/Cas9 system.Science and Technology of Food Industry,37(20):230-234(in Chinese)
Morin E,Kohler A,Baker AR,Foulongne-Oriol M,Lombard V,Nagye LG,Ohm RA,Patyshakuliyeva A,Brun A,Aerts AL,Bailey AM,Billette C,Coutinho PM,Deakin G,Doddapaneni H,Floudas D,Grimwood J,Hilden K,Kues U,Labutti KM,Lapidus A,Lindquist EA,Lucas SM,Murat C,Riley RW,Salamov AA,Schmutz J,Subramanian V,Wosten HAB,Xu J,Eastwood DC,Foster GD,Sonnenberg ASM,Cullen D,de Vries RP,Lundell T,Hibbett DS,Henrissat B,Burton KS,Kerrigan RW,Challen MP,Grigoriev IV,Martin F,2012.Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.Proceedings of the National Academy of Sciences,109(43):17501-17506
Park YJ,Baek JH,Lee S,Kim C,Rhee H,Kim H,Seo JS,Park HR,Yoon DE,Nam JY,Kim HI,Kim JG,Yoon H,Kang HW,Cho JY,Song ES,Sung GH,Yoo YB,Lee CS,Lee BM,Kong WS,2014.Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.PLoS One,9(4):e93560
Pattanayak V,Lin S,Guilinger JP,Ma E,Doudna JA,Liu DR,2013.High-throughput profiling of off-target DNAcleavage reveals RNA-programmed Cas9 nuclease specificity.Nature Biotechnology,31(9):839-843
Sun YQ,Zhang JJ,Feng ZY,Chen MJ,Wang H,Chen H,2014.Agrobacterium-mediated transformation of edible fungi.Journal of Edible Fungi,33(1):69-75(in Chinese)
Tangmu TYF,Wang JB,2018.Problems in the industrial production of Flammulina velutipes in China.Edible and Medicinal Mushrooms,26(1):23-25(in Chinese)
Waltz E,2016.Gene-edited CRISPR mushroom escapes USregulation.Nature,532(7599):293
Wang PM,Liu XB,Dai YC,Horak E,Yang ZL,2018.Phylogeny and species delimitation of Flammulina:taxonomic status of winter mushroom(enokitake)in East Asia and new species uncovered from Europe by an integrated approach.Mycological Progress,17:1013-1030
Wei W,Xin H,Roy B,Dai J,Miao Y,Gao G,2014.Heritable genome editing with CRISPR/Cas9 in the silkworm,Bombyx mori.PLoS One,9(7):e101210
Yang XQ,Zheng QW,Kang LZ,Li QJ,Ye ZW,Lin JF,Guo LQ,Wei T,2018.Production of Resveratrol in Flammulina velutipes by combinatorial expression of its biosynthetic genes.Industrial Crops and Products,112:755-761
Zhang LX,Zhao LJ,2014.Study on antioxidant activity of Flammulina velutipes polysaccharide.Southwest China Journal of Agricultural Sciences,27(1):240-243(in Chinese)
Zhou P,Li XS,Ma C,An D,Wang CC,Meng XF,2014.Nutrition and medicinal value of Flammulina velutipes.China Fruit and Vegetable,34(12):44-47(in Chinese)
侯波,郑淑彦,邰丽梅,桑兰,吴素蕊,2013.金针菇营养保健功能及食品加工研究现状.食品研究与开发,34(12):122-126
罗润,林俊芳,郭丽琼,叶志伟,郭天芬,云帆,2016.基于CRISPR/Cas9系统的金针菇基因组编辑载体构建.食品工业科技,37(20):230-234
路伟振,吴蓓丽,赵强,2012.G蛋白偶联受体:七次跨膜结构的超级分子--2012年诺贝尔化学奖简介.自然杂志,34(6):337-343
孙运奇,张津京,冯志勇,陈明杰,汪虹,陈辉,2014.根癌农杆菌介导的食用菌转化研究.食用菌学报,21(1):69-75
唐木田郁夫,王建兵,2018.中国金针菇工厂化生产中的问题.食药用菌,26(1):23-25
张丽霞,赵丽娟,2014.金针菇多糖的抗氧化活性研究.西南农业学报,27(1):240-243
周萍,李新胜,马超,安东,王朝川,孟晓峰,2014.金针菇的营养成分及药用价值.中国果菜,34(12):44-47
Cogoni C,Romano N,Macino G,1994.Suppression of gene expression by homologous transgenes.Antonie van Leeuwenhoek,65(3):205-209
Fu Y,Sander JD,Reyon D,Cascio VM,Joung JK,2014.Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.Nature Biotechnology,32(3):279-284
Ge ZW,Liu XB,Zhao K,Yang ZL,2015.Species diversity of Flammulina in China:new varieties and a new record.Mycosystema,34(4):589-603
Hou B,Zheng SY,Tai LM,Sang L,Wu SR,2013.Research on nutrition health function and food processing of Flammulina velutipes.Food Research and Development,34(12):122-126(in Chinese)
Han F,Kang LZ,Zeng XL,Ye ZW,Guo LQ,Lin JF,2014.Bioproduction of baccatin III,an advanced precursor of paclitaxol,with transgenic Flammulina velutipes expressing the 10-deacetylbaccatin III-10-O-acetyl transferase gene.Journal of the Science of Food and Agriculture,94(12):2376-2383
Katayama T,Tanaka Y,Okabe T,Nakamura H,Fujii W,Kitamoto K,Maruyama J,2016.Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae.Biotechnology Letters,38(4):637-642
Li L,Wright SJ,Krystofova S,Park G,Borkovich KA,2007.Heterotrimeric G protein signaling in filamentous fungi.Annual Review of Microbiology,61:423-452
Lu WZ,Wu BL,Zhao Q,2012.G protein coupled receptors,the magic molecules with seven transmembrane helices:a brief introduction to the nobel prize in chemistry 2012.Chinese Journal of Nature,34(6):337-343(in Chinese)
Luo R,Lin JF,Guo LQ,Ye ZW,Guo TF,Yun F,2016.Construction of Flammulina velutipes genome editing vector by using CRISPR/Cas9 system.Science and Technology of Food Industry,37(20):230-234(in Chinese)
Morin E,Kohler A,Baker AR,Foulongne-Oriol M,Lombard V,Nagye LG,Ohm RA,Patyshakuliyeva A,Brun A,Aerts AL,Bailey AM,Billette C,Coutinho PM,Deakin G,Doddapaneni H,Floudas D,Grimwood J,Hilden K,Kues U,Labutti KM,Lapidus A,Lindquist EA,Lucas SM,Murat C,Riley RW,Salamov AA,Schmutz J,Subramanian V,Wosten HAB,Xu J,Eastwood DC,Foster GD,Sonnenberg ASM,Cullen D,de Vries RP,Lundell T,Hibbett DS,Henrissat B,Burton KS,Kerrigan RW,Challen MP,Grigoriev IV,Martin F,2012.Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.Proceedings of the National Academy of Sciences,109(43):17501-17506
Park YJ,Baek JH,Lee S,Kim C,Rhee H,Kim H,Seo JS,Park HR,Yoon DE,Nam JY,Kim HI,Kim JG,Yoon H,Kang HW,Cho JY,Song ES,Sung GH,Yoo YB,Lee CS,Lee BM,Kong WS,2014.Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.PLoS One,9(4):e93560
Pattanayak V,Lin S,Guilinger JP,Ma E,Doudna JA,Liu DR,2013.High-throughput profiling of off-target DNAcleavage reveals RNA-programmed Cas9 nuclease specificity.Nature Biotechnology,31(9):839-843
Sun YQ,Zhang JJ,Feng ZY,Chen MJ,Wang H,Chen H,2014.Agrobacterium-mediated transformation of edible fungi.Journal of Edible Fungi,33(1):69-75(in Chinese)
Tangmu TYF,Wang JB,2018.Problems in the industrial production of Flammulina velutipes in China.Edible and Medicinal Mushrooms,26(1):23-25(in Chinese)
Waltz E,2016.Gene-edited CRISPR mushroom escapes USregulation.Nature,532(7599):293
Wang PM,Liu XB,Dai YC,Horak E,Yang ZL,2018.Phylogeny and species delimitation of Flammulina:taxonomic status of winter mushroom(enokitake)in East Asia and new species uncovered from Europe by an integrated approach.Mycological Progress,17:1013-1030
Wei W,Xin H,Roy B,Dai J,Miao Y,Gao G,2014.Heritable genome editing with CRISPR/Cas9 in the silkworm,Bombyx mori.PLoS One,9(7):e101210
Yang XQ,Zheng QW,Kang LZ,Li QJ,Ye ZW,Lin JF,Guo LQ,Wei T,2018.Production of Resveratrol in Flammulina velutipes by combinatorial expression of its biosynthetic genes.Industrial Crops and Products,112:755-761
Zhang LX,Zhao LJ,2014.Study on antioxidant activity of Flammulina velutipes polysaccharide.Southwest China Journal of Agricultural Sciences,27(1):240-243(in Chinese)
Zhou P,Li XS,Ma C,An D,Wang CC,Meng XF,2014.Nutrition and medicinal value of Flammulina velutipes.China Fruit and Vegetable,34(12):44-47(in Chinese)
侯波,郑淑彦,邰丽梅,桑兰,吴素蕊,2013.金针菇营养保健功能及食品加工研究现状.食品研究与开发,34(12):122-126
罗润,林俊芳,郭丽琼,叶志伟,郭天芬,云帆,2016.基于CRISPR/Cas9系统的金针菇基因组编辑载体构建.食品工业科技,37(20):230-234
路伟振,吴蓓丽,赵强,2012.G蛋白偶联受体:七次跨膜结构的超级分子--2012年诺贝尔化学奖简介.自然杂志,34(6):337-343
孙运奇,张津京,冯志勇,陈明杰,汪虹,陈辉,2014.根癌农杆菌介导的食用菌转化研究.食用菌学报,21(1):69-75
唐木田郁夫,王建兵,2018.中国金针菇工厂化生产中的问题.食药用菌,26(1):23-25
张丽霞,赵丽娟,2014.金针菇多糖的抗氧化活性研究.西南农业学报,27(1):240-243
周萍,李新胜,马超,安东,王朝川,孟晓峰,2014.金针菇的营养成分及药用价值.中国果菜,34(12):44-47