利用CRISPR/Cas9系统定向编辑水稻SD1基因
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摘要
【目的】半矮秆水稻品种的选育和应用是水稻育种的最重大成果之一。半矮秆品种大多是半矮秆基因SD1(semi-dwarf1)功能缺失突变体,为了获得sd1突变体,本研究对SD1基因进行了定向编辑。【方法】利用CRISPR/Cas9系统,以SD1基因为靶基因,构建基因编辑载体CRISPR-SD1,用农杆菌介导的方法转化水稻恢复系申繁17和申繁24。【结果】在2个转化受体的T_0代均获得了纯合的sd1突变体,并且在T_1代株系中分离出了不含转基因序列的植株。2个品种的sd1突变体与各自的野生型相比,株高分别下降了25%左右。【结论】利用CRISPR/Cas9系统可以有效地对目的基因进行编辑,在水稻分子育种领域具有巨大的应用价值。
【Objective】In the last century, the usage of semi-dwarf rice varieties brought about significant improvements and great achievements in rice breeding. Most of the semi-dwarf rice varieties have loss-of-function mutations in the Semi-Dwarf1(SD1) gene. In order to obtain sd1 mutant, targeted editing of SD1 gene was performed. 【Method】SD1 gene editing vector CRISPR-SD1 was constructed by using CRISPR/Cas9 system, and transformed into two rice restore lines Shenfan 17 and Shenfan 24 by the Agrobacterium-mediated method.【Result】 Homozygous sd1 mutants were obtained in T_0 generation in both varieties, and vector-free sd1 mutant lines were segregated from the T_1 population. The plant heights of the two sd1 mutants were decreased by 25% as compared with those of their wild types. 【Conclusion】CRISPR/Cas9 is a powerful tool for rice target gene editing, and has enormous potential in rice molecular breeding.
【Objective】In the last century, the usage of semi-dwarf rice varieties brought about significant improvements and great achievements in rice breeding. Most of the semi-dwarf rice varieties have loss-of-function mutations in the Semi-Dwarf1(SD1) gene. In order to obtain sd1 mutant, targeted editing of SD1 gene was performed. 【Method】SD1 gene editing vector CRISPR-SD1 was constructed by using CRISPR/Cas9 system, and transformed into two rice restore lines Shenfan 17 and Shenfan 24 by the Agrobacterium-mediated method.【Result】 Homozygous sd1 mutants were obtained in T_0 generation in both varieties, and vector-free sd1 mutant lines were segregated from the T_1 population. The plant heights of the two sd1 mutants were decreased by 25% as compared with those of their wild types. 【Conclusion】CRISPR/Cas9 is a powerful tool for rice target gene editing, and has enormous potential in rice molecular breeding.
引文
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[23]谷福林,翟虎渠,万建民,张红生.水稻矮秆性状研究及矮源育种利用.江苏农业学报,2003,19(1):48-54.Gu F L,Zhai H Q,Wan L M,Zhang H S.Study on Inheritance of dwarf character and its utilization in rice(Oryza sativa L.)breeding.Jiangsu J Agric Sci,2003,19(1):48-54.(in Chinese with English abstract)
[24]Asano K,Yamasaki M,Takuno S,Miura K,Katagiri S,Ito T,Doi K,Wu J,Ebana K,Matsumoto T,Innan H,Kitano H,Ashikari M,Matsuoka M.Artificial selection for a green revolution gene during japonica rice domestication.Proc Natl Acad Sci USA,2011,108(27):11034-11039.
[25]Yan M,Zhou S R,Xue H W.CRISPR Primer Designer:Design primers for knockout and chromosome imaging CRISPR-Cas system.J Integr Plant Biol,2015,57(7):613-617.
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[27]Harrison M M,Jenkins B V,O’Connor-Giles K M,Wildonger J.A CRISPR view of development.Genes Dev,2014,28(17):1859-1872.
[2]Liu Q,Xu X,Ren X,Fu H,Wu D,Shu Q.Generation and characterization of low phytic acid germplasm in rice(Oryza sativa L.).Theor Appl Genet,2007,114(5):803-814.
[3]Cho S W,Kim S,Kim J M,Kim J S.Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.Nat Biotechnol,2013,31(3):230-232.
[4]Cong L,Ran F A,Cox D,Lin S,Barretto R,Habib N,Hsu P D,Wu X,Jiang W,Marraffini L A,Zhang F.Multiplex genome engineering using CRISPR/Cas systems.Science,2013,339(6121):819-23.
[5]Jinek M,East A,Cheng A,Lin S,Ma E,Doudna J.RNA-programmed genome editing in human cells.Elife,2013,2:e00471.
[6]Mali P,Aach J,Stranges P B,Esvelt K M,Moosburner M,Kosuri S,Yang L,Church G M.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nat Biotechnol,2013,31(9):833-838.
[7]Mani M,Kandavelou K,Dy F J,Durai S,Chandrasegaran S.Design,engineering,and characterization of zinc finger nucleases.Biochem Biophys Res Commun,2005,335(2):447-457.
[8]Bogdanove A J,Voytas D F.TAL effectors:Customizable proteins for DNA targeting.Science,2011,333(6051):1843-1846.
[9]Samanta M K,Dey A,Gayen S.CRISPR/Cas9:An advanced tool for editing plant genomes.Transgenic Res,2016,25(5):561-573.
[10]Mao Y,Zhang H,Xu N,Zhang B,Gou F,Zhu J K.Application of the CRISPR-Cas system for efficient genome engineering in plants.Mol Plant,2013,6(6):2008-2011.
[11]Shan Q,Wang Y,Li J,Zhang Y,Chen K,Liang Z,Zhang K,Liu J,Xi J J,Qiu J L,Gao C.Targeted genome modification of crop plants using a CRISPR-Cas system.Nat Biotechnol,2013,31(8):686-688.
[12]Jiang W Z,Zhou H B,Bi H H,Fromm M,Yang B,Weeks D P.Demonstration of CRISPR/Cas9/sg RNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice.Nucleic Acids Res,2013,41(20):e188.
[13]Liang Z,Zhang K,Chen K,Gao C.Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.J Genet Genom,2014,41(2):63-68.
[14]Wang Y,Cheng X,Shan Q,Zhang Y,Liu J,Gao C,Qiu JL.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat Biotechnol,2014,32(9):947-951.
[15]Jacobs T B,La Fayette P R,Schmitz R J,Parrott W A.Targeted genome modifications in soybean with CRISPR/Cas9.BMC Biotechnol,2015,15:16.
[16]Li Z,Liu Z B,Xing A,Moon B P,Koellhoffer J P,Huang L,Ward R T,Clifton E,Falco S C,Cigan A M.Cas9-Guide RNA directed genome editing in soybean.Plant Physiol,2015,169(2):960-970.
[17]Sun X,Hu Z,Chen R,Jiang Q,Song G,Zhang H,Xi Y.Targeted mutagenesis in soybean using the CRISPR-Cas9system.Sci Rep,2015,5:10342.
[18]Brooks C,Nekrasov V,Lippman Z B,Van Eck J.Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system.Plant Physiol,2014,166(3):1292-1297.
[19]Zhang H,Zhang J,Wei P,Zhang B,Gou F,Feng Z,Mao Y,Yang L,Zhang H,Xu N,Zhu J K.The CRISPR/Cas9system produces specific and homozygous targeted gene editing in rice in one generation.Plant Biotechnol J,2014,12(6):797-807.
[20]Monna L,Kitazawa N,Yoshino R,Suzuki J,Masuda H,Maehara Y,Tanji M,Sato M,Nasu S,Minobe Y.Positional cloning of rice semidwarfing gene,sd-1:Rice“Green Revolution Gene”encodes a mutant enzyme involved in gibberellin synthesis.DNA Res,2002,9(1):11-17.
[21]Spielmeyer W,Ellis M H,Chandler P M.Semidwarf(sd-1),“green revolution”rice,contains a defective gibberellin 20-oxidase gene.Proc Natl Acad Sci USA,2002,99(13):9043-9048.
[22]Sasaki A,Ashikari M,Ueguchi-Tanaka M,Itoh H,Nishimura A,Swapan D,Ishiyama K,Saito T,Kobayashi M,Khush G S,Kitano H,Matsuoka M.Green revolution:A mutant gibberellin-synthesis gene in rice.Nature,2002,416(6882):701-702.
[23]谷福林,翟虎渠,万建民,张红生.水稻矮秆性状研究及矮源育种利用.江苏农业学报,2003,19(1):48-54.Gu F L,Zhai H Q,Wan L M,Zhang H S.Study on Inheritance of dwarf character and its utilization in rice(Oryza sativa L.)breeding.Jiangsu J Agric Sci,2003,19(1):48-54.(in Chinese with English abstract)
[24]Asano K,Yamasaki M,Takuno S,Miura K,Katagiri S,Ito T,Doi K,Wu J,Ebana K,Matsumoto T,Innan H,Kitano H,Ashikari M,Matsuoka M.Artificial selection for a green revolution gene during japonica rice domestication.Proc Natl Acad Sci USA,2011,108(27):11034-11039.
[25]Yan M,Zhou S R,Xue H W.CRISPR Primer Designer:Design primers for knockout and chromosome imaging CRISPR-Cas system.J Integr Plant Biol,2015,57(7):613-617.
[26]Qiao F,Zhao K J.The Influence of RNAi targeting of Os GA20ox2 gene on plant height in rice.Plant Mol Biol Rep,2011,29(4):952.
[27]Harrison M M,Jenkins B V,O’Connor-Giles K M,Wildonger J.A CRISPR view of development.Genes Dev,2014,28(17):1859-1872.