基于CRISPR-Cas9基因编辑技术创制大豆gmnark超结瘤突变体
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摘要
为研究豆科作物结瘤自我调节机制(autoregulation of nodulation, AON)的作用机理,运用CRISPR-Cas9基因编辑技术创制大豆品种华春6号超结瘤gmnark突变体,设计3条靶向目的基因GmNARK的特异sgRNA,构建CRISPR-Cas9敲除载体。通过毛根转化试验选取sgRNA-B和sgRNA-C两条编辑效率较高的sgRNA用于大豆稳定转化,在T_1代筛选出8种不同突变类型的突变体,在T_2代通过营养液水培试验证明其中5种突变体有超结瘤的表型。选取gmnark-A突变体作进一步表型分析,发现其具有超结瘤、植株矮小和叶片深绿的表型。本研究所创制的gmnark突变体是研究AON途径作用机制与根瘤发育的重要遗传材料,同时此新种质资源具有作为"绿肥"与其它作物进行间作或轮作的巨大潜力。
In order to study the mechanism of the autoregulation of nodulation(AON) in the legumes, the gene editing technology of CRISPR-Cas9 system was used to create the gmnark mutant of the soybean variety Huachun 6, and three specific sgRNAs were designed to locate the targeted gene GmNARK and constructed into a CRISPR-Cas9 knockout vector. Two sgRNAs with high editing efficiency, sgRNA-B and sgRNA-C, were selected for stable transformation through hairy root transformation experiments. Furthermore, eight mutants of different mutation types were screened in T_1 generation and five of them had the phenotype of supernodulation which is verified by hydroponic experiment. In the further phenotypic analysis of gmnark-A mutant, it was found that gmnark-A owned a larger number of nodules, smaller shoot and root and greener leaves than WT. These data proved that the gmnark mutant created in the study is an important genetic material for studying the mechanism of AON pathway and the development of nodule. At the same time, this new germplasm resource has great potential in intercropping with other crops as ‘green manure'.
In order to study the mechanism of the autoregulation of nodulation(AON) in the legumes, the gene editing technology of CRISPR-Cas9 system was used to create the gmnark mutant of the soybean variety Huachun 6, and three specific sgRNAs were designed to locate the targeted gene GmNARK and constructed into a CRISPR-Cas9 knockout vector. Two sgRNAs with high editing efficiency, sgRNA-B and sgRNA-C, were selected for stable transformation through hairy root transformation experiments. Furthermore, eight mutants of different mutation types were screened in T_1 generation and five of them had the phenotype of supernodulation which is verified by hydroponic experiment. In the further phenotypic analysis of gmnark-A mutant, it was found that gmnark-A owned a larger number of nodules, smaller shoot and root and greener leaves than WT. These data proved that the gmnark mutant created in the study is an important genetic material for studying the mechanism of AON pathway and the development of nodule. At the same time, this new germplasm resource has great potential in intercropping with other crops as ‘green manure'.
引文
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[2] Herridge D F,Peoples M B,Boddey R M.Global inputs of biological nitrogen fixation in agricultural systems[J].Plant Soil,2008,311(1-2):1-18.
[3] Oldroyd G E,Downie J A.Coordinating nodule morphogenesis with rhizobial infection in legumes[J].Annual Review of Plant Biology,2008,59:519-546.
[4] Oldroyd G E,Murray J D,Poole P S,et al.The rules of engagement in the legume-rhizobial symbiosis[J].Annual Review of Genetics,2012,45:119-144.
[5] Brett J F,Arief I,Satomi H,et al.Molecular analysis of legume nodule development and autoregulation[J].Journal of Integrative Plant Biology,2010,52 (1):61-76.
[6] Okamoto S,Shinohara H,Mori T,et al.Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase[J].Nature Communication,2013,4:2191.
[7] Reid D E,Ferguson B J,Gresshoff P M,et al.Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule formation[J].Molecular Plant-Microbe Interactions,2013,24(5):606-618.
[8] Searle I R,Men A E,Laniya T S,et al.Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase[J].Science,2003,299(5603):109-112.
[9] Sasaki T,Suzaki T,Soyano T,et al.Shoot- derived cytokinins systemically regulate root nodulation[J].Nature Communication,2014,5:4983.
[10] Daniela T,Zhe Y,Dennis B,et al.Systemic control of legume susceptibility to rhizobial infection by a mobile microRNA[J].Science,2018,362(6411):233-236.
[11] Takahara M,Magori M,Soyano T,et al.Too much love,a novel Kelch repeat-containing F-box protein,functions in the long- distance regulation of the legume-Rhizobium symbiosis[J].Plant Cell Physiology,2013,54(4):433-447.
[12] Ogawa M,Shinohara H,Sakagai Y,et al.Arabidopsis CLV3 peptide directly binds CLV1 ectodomain[J].Science,2008,319(5861):294.
[13] Ran F A,Hsu P D,Wright J,et al.Genome engineering using the CRISPR/Cas9 system[J].Nature Protocols,2013,8(11):2281-2308.
[14] Feng Z,Zhang B,Ding W,et al.Efficient genome editing in plants using a CRISPR/Cas system[J].Cell Research,2013,23(10):1229-1232.
[15] Ma X L,Zhang Q,Zhu Q,et al.A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicotplants[J].Molecular Plant,2015,8(8):1274-1284.
[16] Burgess.Technology:A CRISPR/Cas9 genome editing tool[J].Nature Reviews Genetics,2013,14(2):80.
[17] Fu Y,Foden J A,Khayter C,et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J].Nature Biotechnology,2013,31(9):822-826.
[18] Miao J,Guo D,Zhang J,et al.Targeted mutagenesis in rice using CRISPR-Cas system[J].Cell Research,2013,23(10):1233-1236.
[19] Duan J,Lu G,Xie Z,et al.Genome-wide identification of CRISPR/Cas9 off-targets in human genome[J].Cell Research,2014,24(8):1009-1012.
[20] Liang Z,Zhang K,Chen K,et al.Targeted mutagenesis in Zea mays using TA1LENs and the CRISPR[J].Journal of Genetics and Genomics,2014,41(2):63-68.
[21] Masafumi M,Toki S,Endo M.Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice[J].Plant Cell Reports,2015,34(10):1807-1815.
[22] Sun Y,Zhang X,Wu C,et al.Engineering herbicide-resistant rice plants through CRISPR/Cas9-rediated homologous recombination of acetolactate synthase[J].Molecular Plant,2016,9(4):628-631.
[23] Jacobs T B,LaFayette P R,Schmitz R J,et al.Targeted genome modifications in soybean with CRISPR/Cas9[J].BMC Biotechnology,2015,15(1):16.
[24] Du H,Zeng X,Zhao M,et al.Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9[J].Journal of Biotechnology,2016,217:90-97.
[25] Kanazashi Y,Hirose A,Takahashi I,et al.Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA[J].Plant Cell Reports,2018,37(3):553-563.
[26] Song S,Hou W,Godo I,et al.Soybean seeds expressing feedback-insensitive cystathionine gamma- synthase exhibit a higher content of methionine[J].Journal of Experimental Botany,2013,64(7),1917-1926.
[27] Cai Y,Chen L,Liu X,et al.CRISPR/Cas9-mediated genome editing in soybean hairy roots[J].PLoS One,2015,10(8):e0136064.
[28] Tang F,Yang S,Liu J,et al.Rj4,a gene controlling nodulation specificity in soybeans,encodes a thaumatin-like protein but not the one previously reported[J].Plant Physiology,2016,170(1):26-32.
[29] Fang Y,Tyler B M.Efficient disruption and replacement of an effector gene in the oomycete P hytophthora sojae using CRISPR/Cas9[J].Molecular Plant Pathology,2016,17(1):127-139.
[30] Cai Y,Chen L,Liu X,et al.CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean[J].Plant Biotechnology Journal,2018,16(1):176-185.
[31] Schmutz J,Cannon S B,Schlueter J,et al.Genome sequence of the palaeopolyploid soybean[J].Nature,2010,463(7278):178-183.
[32] Miyahara A,Hirani T A,Oakes M,et al.Soybean nodule autoregulation receptor kinase phosphorylates two kinase-associated protein phosphatases in vitro[J].Journal of Biological Chemistry,2008,283(37):25381-25391.
[33] Liu L,Lei X L,Huang G Z,et al.Influences of mechanical sowing and transplanting on nitrogen accumulation,distribution and C/N of hybrid rice cultivars[J].Journal of Plant Nutrition and Fertilizer,2014,20(4):831-844.