CRISPR/Cas9技术定点编辑水稻谷蛋白基因GluA
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摘要
通过CRISPR/Cas9基因编辑技术对粳稻品种‘嘉花1号’的谷蛋白基因GluA3(LOC_Os03 g31360)进行编辑获得转基因T0代植株,并对其衍生的T1代28个单株进行鉴定分析。结果表明:获得了两种类型的GluA3编辑突变植株;突变体的未成熟胚乳中GluA3 RNA表达水平明显下调;突变体谷蛋白含量呈下降趋势。对‘嘉花1号’野生型及突变体水稻农艺性状考查发现,突变体水稻穗数和产量下降明显。试验表明,CRISPR/Cas9技术可有效编辑水稻谷蛋白基因,可为水稻谷蛋白品质育种提供理论指导。
The gluten gene GluA3( LOC_Os03 g31360) in japonica rice variety ‘Jiahua 1'was edited by CRISPR/Cas9 technology,and 28 T1 individuals derived from the transgenic T0 generation were identified and analyzed. The results showed that two types of GluA3 edited-mutant plants were obtained; the expression levels of GluA3 RNA in immature endosperm of the two edited-mutants were significantly down-regulated; the gluten content of the mutants had a downward trend. The agronomic traits of ‘Jiahua 1'wild type and mutant rice were also examined. It was found that the panicle number and yield of mutant rice decreased obviously. The research showed that CRISPR/Cas9 technology could edit gluten gene effectively and it would provide theoretical guidance for breeding and improving quality of gluten in rice.
The gluten gene GluA3( LOC_Os03 g31360) in japonica rice variety ‘Jiahua 1'was edited by CRISPR/Cas9 technology,and 28 T1 individuals derived from the transgenic T0 generation were identified and analyzed. The results showed that two types of GluA3 edited-mutant plants were obtained; the expression levels of GluA3 RNA in immature endosperm of the two edited-mutants were significantly down-regulated; the gluten content of the mutants had a downward trend. The agronomic traits of ‘Jiahua 1'wild type and mutant rice were also examined. It was found that the panicle number and yield of mutant rice decreased obviously. The research showed that CRISPR/Cas9 technology could edit gluten gene effectively and it would provide theoretical guidance for breeding and improving quality of gluten in rice.
引文
[1]OGAWA M,KUMAMARA T,SATOH H,et al. Purification of Protein Body-I of Rice Seed and its Polypeptide Composition[J]. Plant Cell,1987,28(8):1517-1527.
[2]RESURRECCION A P,LI X,OKITA T H,et al. Characterization of Poorly Digested Protein of Cooked Rice Protein Bodies[J]. Cereal Chem,1993,70(1):101-104.
[3]李文.水稻谷蛋白囊泡转运及转基因水稻蛋白组学研究[D].武汉:武汉大学,2014.
[4]曲乐庆,魏晓丽,佐藤光,等.水稻种子贮藏谷蛋白α-2亚基减少突变体[J].植物学报,2001,43(11):1167-1171.
[5]张宪银,薛庆中.水稻种子贮藏蛋白分子生物学研究进展[J].浙江农业学报,2000,12(2):57-60.
[6]TANAKA K,SUGIMOTO T,OGAWA M,H,et al. Isolation and Characterization of Two Types of Protein Bodies in the Rice Endosperm[J].Agric. Biol. Chem.,1980,44(7):1633-1639.
[7]KUMAMARU T,SATOH H,IWATA N,et al. Mutants for Rice Storage Proteins[J]. Theor Appl Genet,1988,76:11-16.
[8]王广元,郭艳萍,史通麟,等.水稻13 k Da醇溶谷蛋白b组分缺失的天然突变体[J].山西大学学报,2010,33(S1):77-80.
[9]KUSABA M,MIYAHARA K,IIDA S,et al. Low Glutelin Content 1:A Dominant Mutation that Suppresses the Glutelin Multigene Family via RNA Silencing in Rice[J]. The Plant Cell,2003,15(6):1455-1467.
[10]王才林,田孟祥,陈涛,等.水稻57H突变体glup-t的遗传分析与基因定位[J].作物学报,2011,37(4):717-722.
[11]万建民,牛洪斌,覃怀德,等.水稻谷蛋白的一个新基因克隆及表达分析[J].作物学报,2007,33(3):349-355.
[12]TAKAIWA F,OONO K. Genomic DNA Sequences of Two New Genes for New Storage Protein Glutelin in Rice[J]. Jpn. J. Genet.,1991,66(2):161-171.
[13]田孟祥,陈涛,张亚东,等.水稻谷蛋白突变体的分类及其分子生物学研究进展[J].江苏农业学报,2010,26(4):874-881.
[14]万建民,翟虎渠,刘世家,等.功能性专用水稻品种W3660的选育[J].作物杂志,2004(5):58.
[15]NISHIMURA M,KUSABA M,MIYAHARA K,et al. New Rice Varieties with Low Levels of Easy-to-digest Protein,‘LGC-Katsu’and‘LGCJun’[J]. Breeding Science,2005,55(1):103-105.
[16]田孟祥,陈涛,张亚东,等.两个低谷蛋白基因插入缺失标记的设计与验证[J].分子植物育种,2010,8(2):340-344.
[17]MA X L,ZHAN Q Y,ZHU Q L,et al. A Robust CRISPR/Cas9 System for Convenient,High-efficiency Multiplex Genome Editing in monocot and dicot Plants[J]. Molecular Plant,2015,8:1274-1284.
[18]MURRAYM G,THOMPSON W F. Rapid Isolation of High Molecular Weight Plant DNA[J]. Nucleic Acids Research,1980,8:4321-4326.
[19]张建辉,李小艳,林冬枝,等.一个新的水稻类病斑突变体的鉴定及其基因分子定位[J].基因组学与应用生物学,2011,30(8):1284-1289.
[20]邵高能,谢黎虹,焦桂爱,等.利用CRISPR/CAS9技术编辑水稻香味基因Badh2[J].中国水稻科学,2017,31(2):216-222.
[21]尹祥佳,李艳玫,郝楠. CRISPR/Cas9基因编辑系统及其在水稻育种中的应用综述[J].甘肃农业科技,2017(10):80-84.
[22]沈兰,李健,付亚萍,等.利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状[J].中国水稻科学,2017,31(3):223-231.
[23]周文甲,田晓杰,任月坤,等.利用CRISPR/Cas9创造早熟香味水稻[J].土壤与作物,2017,6(2):146-152.
[24]赵丰兰,段永波,盛玮,等.基于降低水稻谷蛋白多成员表达水平的干涉表达载体构建[J].甘肃农业科技,2015(9):28-31.
[2]RESURRECCION A P,LI X,OKITA T H,et al. Characterization of Poorly Digested Protein of Cooked Rice Protein Bodies[J]. Cereal Chem,1993,70(1):101-104.
[3]李文.水稻谷蛋白囊泡转运及转基因水稻蛋白组学研究[D].武汉:武汉大学,2014.
[4]曲乐庆,魏晓丽,佐藤光,等.水稻种子贮藏谷蛋白α-2亚基减少突变体[J].植物学报,2001,43(11):1167-1171.
[5]张宪银,薛庆中.水稻种子贮藏蛋白分子生物学研究进展[J].浙江农业学报,2000,12(2):57-60.
[6]TANAKA K,SUGIMOTO T,OGAWA M,H,et al. Isolation and Characterization of Two Types of Protein Bodies in the Rice Endosperm[J].Agric. Biol. Chem.,1980,44(7):1633-1639.
[7]KUMAMARU T,SATOH H,IWATA N,et al. Mutants for Rice Storage Proteins[J]. Theor Appl Genet,1988,76:11-16.
[8]王广元,郭艳萍,史通麟,等.水稻13 k Da醇溶谷蛋白b组分缺失的天然突变体[J].山西大学学报,2010,33(S1):77-80.
[9]KUSABA M,MIYAHARA K,IIDA S,et al. Low Glutelin Content 1:A Dominant Mutation that Suppresses the Glutelin Multigene Family via RNA Silencing in Rice[J]. The Plant Cell,2003,15(6):1455-1467.
[10]王才林,田孟祥,陈涛,等.水稻57H突变体glup-t的遗传分析与基因定位[J].作物学报,2011,37(4):717-722.
[11]万建民,牛洪斌,覃怀德,等.水稻谷蛋白的一个新基因克隆及表达分析[J].作物学报,2007,33(3):349-355.
[12]TAKAIWA F,OONO K. Genomic DNA Sequences of Two New Genes for New Storage Protein Glutelin in Rice[J]. Jpn. J. Genet.,1991,66(2):161-171.
[13]田孟祥,陈涛,张亚东,等.水稻谷蛋白突变体的分类及其分子生物学研究进展[J].江苏农业学报,2010,26(4):874-881.
[14]万建民,翟虎渠,刘世家,等.功能性专用水稻品种W3660的选育[J].作物杂志,2004(5):58.
[15]NISHIMURA M,KUSABA M,MIYAHARA K,et al. New Rice Varieties with Low Levels of Easy-to-digest Protein,‘LGC-Katsu’and‘LGCJun’[J]. Breeding Science,2005,55(1):103-105.
[16]田孟祥,陈涛,张亚东,等.两个低谷蛋白基因插入缺失标记的设计与验证[J].分子植物育种,2010,8(2):340-344.
[17]MA X L,ZHAN Q Y,ZHU Q L,et al. A Robust CRISPR/Cas9 System for Convenient,High-efficiency Multiplex Genome Editing in monocot and dicot Plants[J]. Molecular Plant,2015,8:1274-1284.
[18]MURRAYM G,THOMPSON W F. Rapid Isolation of High Molecular Weight Plant DNA[J]. Nucleic Acids Research,1980,8:4321-4326.
[19]张建辉,李小艳,林冬枝,等.一个新的水稻类病斑突变体的鉴定及其基因分子定位[J].基因组学与应用生物学,2011,30(8):1284-1289.
[20]邵高能,谢黎虹,焦桂爱,等.利用CRISPR/CAS9技术编辑水稻香味基因Badh2[J].中国水稻科学,2017,31(2):216-222.
[21]尹祥佳,李艳玫,郝楠. CRISPR/Cas9基因编辑系统及其在水稻育种中的应用综述[J].甘肃农业科技,2017(10):80-84.
[22]沈兰,李健,付亚萍,等.利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状[J].中国水稻科学,2017,31(3):223-231.
[23]周文甲,田晓杰,任月坤,等.利用CRISPR/Cas9创造早熟香味水稻[J].土壤与作物,2017,6(2):146-152.
[24]赵丰兰,段永波,盛玮,等.基于降低水稻谷蛋白多成员表达水平的干涉表达载体构建[J].甘肃农业科技,2015(9):28-31.
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