CRISPR/Cas9系统编辑水稻Wx基因
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摘要
【目的】直链淀粉含量与稻米品质密切相关。Wx基因是控制水稻直链淀粉合成的主效基因,通过对Wx基因定点编辑以获得稳定遗传、直链淀粉含量适宜的突变体。【方法】构建CRISPR/Cas9表达载体p GK03-Wx-g RNA(靶点1和2分别在Wx基因第1和第2外显子),利用工程菌EHA105遗传转化超级稻楚粳27,潮霉素筛选获得转化株系,对转化株系及其后代进行分子检测、测序、基因表达和遗传稳定性分析以及直链淀粉含量测定。【结果】获得9个独立的T_0代转化株系,靶点1(L1~L5)5个株系,突变频率100%,靶点2(L6~L9)4个株系,突变频率75%。由T_0代突变体衍生出T_1和T_2代株系,测序发现T_0、T_1和T_2代株系出现缺失(单、双、多碱基缺失)和单碱基插入两种突变类型;T_0至T_1代部分株系(L1、L2、L3和L6)发生再编辑,T_1至T_2代遗传稳定。与野生型相比,突变株系RNA水平Wx基因表达量显著下降(P<0.01),稻米直链淀粉含量显著降低(P<0.01),从17.5%降到1.93%。【结论】利用CRISPR/Cas9系统成功编辑水稻Wx基因,获得了稳定遗传、低直链淀粉含量的突变体,为稻米品质改良提供了材料。
【Objective】Amylose content is closely related to rice quality. The objective of this research is to knockout Wx gene for obtaining rice mutants with suitable amylose content.【Method】Vector p GK03-Wx-g RNA, a CRISPR/Cas9 system, targeted either the first(target 1) or the second(target 2) exon of Wx gene was constructed and transformed into super-rice Chujing 27 to edit the Wx gene. Independent transformants were obtained via Agrobacterium-mediated transformation with hygromycin selection. The transgenic lines of T_0, T_1 and T_2 generations were identified by DNA sequencing, in addition to q PCR and amylose content assay of T_1. 【Result】Nine T_0 independent transgenic lines were generated, the L1-L5 from target 1 and L6-L9 from target 2. Eight lines(L1-L8) were mutants and L9 was not confirmed as mutant by sequencing. Therefore, the mutation rate was 100% for target 1, and 75% for target 2. T_1 and T_2 generations of the eight lines(L1-L8) were further sequenced, which revealed two types of mutation: base deletion(single base, double and up to fourteen bases), and base insertion. Some lines(L1, L2, L3 and L6) were reedited at T_1 generation when compared to its T_0 generation, while the rest lines were stable at the mutation sites. All the eight mutants were stable when comparing their target gene sequence at T_1 and T_2 generations. q PCR results indicated that Wx gene expression at RNA level was considerably reduced in the mutant. The seed amylose content was reduced from 17.5% to 1.93%(P<0.01). 【Conclusion】The Wx gene was successfully edited by CRISPR/Cas9 system in rice, the inheritable mutants with average amylose content at 1.93% were obtained, which would provide valuable germplasm for rice quality improvement.
【Objective】Amylose content is closely related to rice quality. The objective of this research is to knockout Wx gene for obtaining rice mutants with suitable amylose content.【Method】Vector p GK03-Wx-g RNA, a CRISPR/Cas9 system, targeted either the first(target 1) or the second(target 2) exon of Wx gene was constructed and transformed into super-rice Chujing 27 to edit the Wx gene. Independent transformants were obtained via Agrobacterium-mediated transformation with hygromycin selection. The transgenic lines of T_0, T_1 and T_2 generations were identified by DNA sequencing, in addition to q PCR and amylose content assay of T_1. 【Result】Nine T_0 independent transgenic lines were generated, the L1-L5 from target 1 and L6-L9 from target 2. Eight lines(L1-L8) were mutants and L9 was not confirmed as mutant by sequencing. Therefore, the mutation rate was 100% for target 1, and 75% for target 2. T_1 and T_2 generations of the eight lines(L1-L8) were further sequenced, which revealed two types of mutation: base deletion(single base, double and up to fourteen bases), and base insertion. Some lines(L1, L2, L3 and L6) were reedited at T_1 generation when compared to its T_0 generation, while the rest lines were stable at the mutation sites. All the eight mutants were stable when comparing their target gene sequence at T_1 and T_2 generations. q PCR results indicated that Wx gene expression at RNA level was considerably reduced in the mutant. The seed amylose content was reduced from 17.5% to 1.93%(P<0.01). 【Conclusion】The Wx gene was successfully edited by CRISPR/Cas9 system in rice, the inheritable mutants with average amylose content at 1.93% were obtained, which would provide valuable germplasm for rice quality improvement.
引文
[1]Hsu Y C,Tseng M C,Wu Y P,Lin M Y,Wei F J,Hwu K K,Hsing Y I,Lin Y R.Genetic factors responsible for eating and cooking qualities of rice grains in a recombinant inbred population of an inter-subspecific cross.Mol Breeding,2014,34(2):655-673.
[2]Wani A A,Singh P,Shah M A,Schweiggert W U,Gul K,Idrees A.Rice starch diversity:Effects on structural,morphological,thermal,and physicochemical properties.Compr Rev Food Sci F,2012,11(5):417-436.
[3]Sano Y.Differential regulation of waxy gene expression in rice endosperm.Theor Appl Genet,1984,68(5):467-473.
[4]Wang Z Y,Zheng F Q,Shen G Z,Gao J P,Snustad D P,Li M G,Zhang J L,Hong M M.The amylose content in rice endosperm is related to the post-transcriptional regulation of the waxy gene.Plant J Cell Mol B,1995,7(4):613-622.
[5]陈雅玲,包劲松.水稻胚乳淀粉合成相关酶的结构、功能及其互作研究进展.中国水稻科学,2017,31(1):1-12.Chen Y L,Bao J S.Progress in structures,functions and interactions of starch synthesis related enzymes in rice endosperm.Chin J Rice Sci,2017,31(1):1-12.(in Chinese with English abstract)
[6]Frances H,Bligh J,Larkin P D,Roach P S,Jones C A,Fu H,Park W D.Use of alternate splice sites in granule-bound starch synthase m RNA from low-amylose rice varieties.Plant Mol Biol,1998,38(3):407-415.
[7]Mikami I,Aikawa M,Hirano H Y,Sano Y.Altered tissue-specific expression at the Wx gene of the opaque mutants in rice.Euphytica,1999,105(2):91-97.
[8]Liu L,Ma X,Liu S,Zhu C,Ling J,Yi H W,Yi S,Yu L R,Hui D,Liang C,Xi L,Zhi Z,Hu Z,Jian W.Identification and characterization of a novel Waxy allele from a Yunnan rice landrace.Plant Mol Biol,2009,71(6):609-626.
[9]Sato H,Suzuli Y,Sakai M,Lmbe T.Molecular characterization of Wx-mq,a novel mutant gene for low-amylose content in endosperm of rice(Oryza sativa L.).Breeding Sci,2002,52(2):131-135.
[10]Larkin P D,Park W D.Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice(Oryza sativa L.).Mol Breeding,2003,12(4):335-339.
[11]Bergman C J,Delgado J T,Mc Clung A M,Fjellstrom R G.An improved method for using a microsatellite in the rice Waxy gene to determine amylose class.Cereal Chem,2001,78(3):257-260.
[12]Inukai T,Sako A,Hirano H Y,Sano Y.Analysis of intragenic recombination at wx in rice:Correlation between the molecular and genetic maps within the locus.Genome,2000,43(4):589-596.
[13]Shen B,Zhang J,Wu H,Ma K,Li Z,Zhang X,Zhang P,Huang X.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting.Cell Res,2013,23(5):720-723.
[14]Jinek M,Chylinski K,Fonfara I,Hauer M,Doudna J A,Charpentier E.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[15]Shan Q W,Wang Y P,Li J,Zhang Y,Chen K L,Liang Z,Zhang K,Liu J X,Xi J J,Qiu J L,Gao C X.Targeted genome modification of crop plants using a CRISPR-Cas system.Nat Biotechnol,2013,31(8):686-688.
[16]Feng Z,Zhang B,Ding W,Liu X,Yang D L,Wei P,Cao F,Zhu S,Zhang F,Mao Y,Zhu J K.Efficient genome editing in plants using a CRISPR/Cas system.Cell Res,2013,23(10):1229-1232.
[17]李开斌,张天春,阮文忠,徐加平,王正伟,黄文兴.超级粳稻楚粳27号的选育及应用.中国稻米,2012,18(5):71-72.Li K B,Zhang T C,Ruan W Z,Xu J P,Wang Z W,Huang W X.Breeding and application of super japonica rice variety Chujing 27.China Rice,2012,18(5):71-72.(in Chinese with English abstract).
[18]曾千春,李旭刚,马炳田,陈松彪,徐鸿林,孟昆,魏晓丽,朱祯.有效去除农杆菌和籼稻转化系统优化.分子植物育种,2003,1(5):783-790.Zeng Q C,Li X G,Ma B T,Chen S B,Xu H L,Meng K,Wei X L,Zhu Z.Efficient elimination of A.tumefaciens and optimization of Agrobacterium-mediated transformation of indica rice.Mol Plant Breed,2003,1(5):783-790.(in Chinese with English abstract)
[19]Zhang G H,Gao M G,Zhang G Z,Sun J J,Jin X M,Wang C Y,Zhao Y,Li S S.A high through-put protocol of plant genomic DNA preparation for PCR.Acta Agron Sin,2013,39(7):1200-1205.
[20]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2[-Delta Delta C(T)]method.Methods,2001,25(4):402-408.
[21]齐万清,蒋刚生,谢建华,秦宣才,李铮友,曾千春.杂交早粳稻米品质在四个生态点的变化.西南农业学报,2008,21(6):1500-1504.Qi W Q,Jiang G S,Xie J H,Qin X C,Li Z Y,Zeng Q C.Quality variation of early japonica hybrid rice from four ecological sites.Southwest China J Agric Sci,2008,21(6):1500-1504.(in Chinese with English abstract)
[22]Xu R F,Li H,Qin R Y,Li J,Qiu C H,Yang Y C,Ma H,Li L,Wei P C,Yang J B.Generation of inheritable and“transgene clean”targeted genome-modified rice in later generations using the CRISPR/Cas9 system.Sci Rep-UK,2015,5:11491.
[23]Mussolino C,Cathomen T.RNA guides genome engineering.Nat Biotechnol,2013,31(3):208-209.
[24]Miao J,Guo D,Zhang J,Huang Q,Qin G,Zhang X,Gu H,Qu L J.Targeted mutagenesis in rice using CRISPRCas system.Cell Res,2013,23(10):1233-1236.
[25]Shan Q W,Wang Y,Li J,Gao C X.Genome editing in rice and wheat using the CRISPR/Cas system.Nat Protoc,2014,9(10):2395-2410.
[26]邵高能,谢黎虹,焦桂爱,魏祥进,圣忠华,唐绍清,胡培松.利用CRISPR/CAS9技术编辑水稻香味基因Badh2.中国水稻科学,2017,31(2):216-222.Shao G N,Xie L H,Jiao G A,Wei X J,Sheng Z H,Tang S Q,Hu P S.CRISPR/CAS9-mediated editing of the fragrant gene Badh2 in rice.Chin J Rice Sci,2017,31(2):216-222.(in Chinese with English abstract)
[27]Ma X L,Zhang Q Y,Zhu Q L,Liu W,Chen Y,Qiu R,Wang B,Yang Z F,Li H Y,Lin Y R,Xie Y Y,Shen R X,Chen S F,Wang Z,Chen Y L,Guo J X,Chen L T,Zhao X C,Dong Z C,Liu Y G.A Robust CRISPR/Cas9system for convenient,high-efficiency multiplex genome editing in Monocot and Dicot plants.Mol Plant,2015,8(8):1274-1284.
[28]Terada R,Nakajima M,Isshiki M,Okagaki R J,Wessler S R,Shimamoto K.Antisense Waxy genes with highly active promoters effectively suppress Waxy gene expression in transgenic rice.Plant Cell Physiol,2000,41(7):881-888.
[29]刘巧泉,王兴稳,陈秀花,王宗阳,汤述翥,洪孟明,顾铭洪.转反义Wx基因糯稻的显性遗传及对稻米粒重的效应分析.中国农业科学,2002,35(2):117-122.Liu Q Q,Wang X W,Chen X H,Wang Z Y,Tang S Z,Hong M M,Gu M H.Effect of dominant Waxy character on kernel weight of transgenic rice with antisense Wx gene.Sci Agric Sin,2002,35(2):117-122.(in Chinese with English abstract)
[30]郭健.应用RNA干涉技术改变水稻淀粉组成的研究.广州:华南农业大学,2008.Guo J.Improving the composition of rice with RNA interference technology.Guangzhou:South China Agricultural University,2008.(in Chinese with English abstract)
[31]Tian Z X,Qian Q,Liu Q Q,Yan M X,Liu X F,Yan C J,Liu G F,Gao Z Y,Tang S Z,Zeng D L,Wang Y H,Yu J M,Gu M H,Li J Y.Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities.Proc Natl Acad Sci USA,2009,106(51):21760-21765.
[2]Wani A A,Singh P,Shah M A,Schweiggert W U,Gul K,Idrees A.Rice starch diversity:Effects on structural,morphological,thermal,and physicochemical properties.Compr Rev Food Sci F,2012,11(5):417-436.
[3]Sano Y.Differential regulation of waxy gene expression in rice endosperm.Theor Appl Genet,1984,68(5):467-473.
[4]Wang Z Y,Zheng F Q,Shen G Z,Gao J P,Snustad D P,Li M G,Zhang J L,Hong M M.The amylose content in rice endosperm is related to the post-transcriptional regulation of the waxy gene.Plant J Cell Mol B,1995,7(4):613-622.
[5]陈雅玲,包劲松.水稻胚乳淀粉合成相关酶的结构、功能及其互作研究进展.中国水稻科学,2017,31(1):1-12.Chen Y L,Bao J S.Progress in structures,functions and interactions of starch synthesis related enzymes in rice endosperm.Chin J Rice Sci,2017,31(1):1-12.(in Chinese with English abstract)
[6]Frances H,Bligh J,Larkin P D,Roach P S,Jones C A,Fu H,Park W D.Use of alternate splice sites in granule-bound starch synthase m RNA from low-amylose rice varieties.Plant Mol Biol,1998,38(3):407-415.
[7]Mikami I,Aikawa M,Hirano H Y,Sano Y.Altered tissue-specific expression at the Wx gene of the opaque mutants in rice.Euphytica,1999,105(2):91-97.
[8]Liu L,Ma X,Liu S,Zhu C,Ling J,Yi H W,Yi S,Yu L R,Hui D,Liang C,Xi L,Zhi Z,Hu Z,Jian W.Identification and characterization of a novel Waxy allele from a Yunnan rice landrace.Plant Mol Biol,2009,71(6):609-626.
[9]Sato H,Suzuli Y,Sakai M,Lmbe T.Molecular characterization of Wx-mq,a novel mutant gene for low-amylose content in endosperm of rice(Oryza sativa L.).Breeding Sci,2002,52(2):131-135.
[10]Larkin P D,Park W D.Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice(Oryza sativa L.).Mol Breeding,2003,12(4):335-339.
[11]Bergman C J,Delgado J T,Mc Clung A M,Fjellstrom R G.An improved method for using a microsatellite in the rice Waxy gene to determine amylose class.Cereal Chem,2001,78(3):257-260.
[12]Inukai T,Sako A,Hirano H Y,Sano Y.Analysis of intragenic recombination at wx in rice:Correlation between the molecular and genetic maps within the locus.Genome,2000,43(4):589-596.
[13]Shen B,Zhang J,Wu H,Ma K,Li Z,Zhang X,Zhang P,Huang X.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting.Cell Res,2013,23(5):720-723.
[14]Jinek M,Chylinski K,Fonfara I,Hauer M,Doudna J A,Charpentier E.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[15]Shan Q W,Wang Y P,Li J,Zhang Y,Chen K L,Liang Z,Zhang K,Liu J X,Xi J J,Qiu J L,Gao C X.Targeted genome modification of crop plants using a CRISPR-Cas system.Nat Biotechnol,2013,31(8):686-688.
[16]Feng Z,Zhang B,Ding W,Liu X,Yang D L,Wei P,Cao F,Zhu S,Zhang F,Mao Y,Zhu J K.Efficient genome editing in plants using a CRISPR/Cas system.Cell Res,2013,23(10):1229-1232.
[17]李开斌,张天春,阮文忠,徐加平,王正伟,黄文兴.超级粳稻楚粳27号的选育及应用.中国稻米,2012,18(5):71-72.Li K B,Zhang T C,Ruan W Z,Xu J P,Wang Z W,Huang W X.Breeding and application of super japonica rice variety Chujing 27.China Rice,2012,18(5):71-72.(in Chinese with English abstract).
[18]曾千春,李旭刚,马炳田,陈松彪,徐鸿林,孟昆,魏晓丽,朱祯.有效去除农杆菌和籼稻转化系统优化.分子植物育种,2003,1(5):783-790.Zeng Q C,Li X G,Ma B T,Chen S B,Xu H L,Meng K,Wei X L,Zhu Z.Efficient elimination of A.tumefaciens and optimization of Agrobacterium-mediated transformation of indica rice.Mol Plant Breed,2003,1(5):783-790.(in Chinese with English abstract)
[19]Zhang G H,Gao M G,Zhang G Z,Sun J J,Jin X M,Wang C Y,Zhao Y,Li S S.A high through-put protocol of plant genomic DNA preparation for PCR.Acta Agron Sin,2013,39(7):1200-1205.
[20]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2[-Delta Delta C(T)]method.Methods,2001,25(4):402-408.
[21]齐万清,蒋刚生,谢建华,秦宣才,李铮友,曾千春.杂交早粳稻米品质在四个生态点的变化.西南农业学报,2008,21(6):1500-1504.Qi W Q,Jiang G S,Xie J H,Qin X C,Li Z Y,Zeng Q C.Quality variation of early japonica hybrid rice from four ecological sites.Southwest China J Agric Sci,2008,21(6):1500-1504.(in Chinese with English abstract)
[22]Xu R F,Li H,Qin R Y,Li J,Qiu C H,Yang Y C,Ma H,Li L,Wei P C,Yang J B.Generation of inheritable and“transgene clean”targeted genome-modified rice in later generations using the CRISPR/Cas9 system.Sci Rep-UK,2015,5:11491.
[23]Mussolino C,Cathomen T.RNA guides genome engineering.Nat Biotechnol,2013,31(3):208-209.
[24]Miao J,Guo D,Zhang J,Huang Q,Qin G,Zhang X,Gu H,Qu L J.Targeted mutagenesis in rice using CRISPRCas system.Cell Res,2013,23(10):1233-1236.
[25]Shan Q W,Wang Y,Li J,Gao C X.Genome editing in rice and wheat using the CRISPR/Cas system.Nat Protoc,2014,9(10):2395-2410.
[26]邵高能,谢黎虹,焦桂爱,魏祥进,圣忠华,唐绍清,胡培松.利用CRISPR/CAS9技术编辑水稻香味基因Badh2.中国水稻科学,2017,31(2):216-222.Shao G N,Xie L H,Jiao G A,Wei X J,Sheng Z H,Tang S Q,Hu P S.CRISPR/CAS9-mediated editing of the fragrant gene Badh2 in rice.Chin J Rice Sci,2017,31(2):216-222.(in Chinese with English abstract)
[27]Ma X L,Zhang Q Y,Zhu Q L,Liu W,Chen Y,Qiu R,Wang B,Yang Z F,Li H Y,Lin Y R,Xie Y Y,Shen R X,Chen S F,Wang Z,Chen Y L,Guo J X,Chen L T,Zhao X C,Dong Z C,Liu Y G.A Robust CRISPR/Cas9system for convenient,high-efficiency multiplex genome editing in Monocot and Dicot plants.Mol Plant,2015,8(8):1274-1284.
[28]Terada R,Nakajima M,Isshiki M,Okagaki R J,Wessler S R,Shimamoto K.Antisense Waxy genes with highly active promoters effectively suppress Waxy gene expression in transgenic rice.Plant Cell Physiol,2000,41(7):881-888.
[29]刘巧泉,王兴稳,陈秀花,王宗阳,汤述翥,洪孟明,顾铭洪.转反义Wx基因糯稻的显性遗传及对稻米粒重的效应分析.中国农业科学,2002,35(2):117-122.Liu Q Q,Wang X W,Chen X H,Wang Z Y,Tang S Z,Hong M M,Gu M H.Effect of dominant Waxy character on kernel weight of transgenic rice with antisense Wx gene.Sci Agric Sin,2002,35(2):117-122.(in Chinese with English abstract)
[30]郭健.应用RNA干涉技术改变水稻淀粉组成的研究.广州:华南农业大学,2008.Guo J.Improving the composition of rice with RNA interference technology.Guangzhou:South China Agricultural University,2008.(in Chinese with English abstract)
[31]Tian Z X,Qian Q,Liu Q Q,Yan M X,Liu X F,Yan C J,Liu G F,Gao Z Y,Tang S Z,Zeng D L,Wang Y H,Yu J M,Gu M H,Li J Y.Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities.Proc Natl Acad Sci USA,2009,106(51):21760-21765.