油菜乙酰乳酸合成酶抑制剂类除草剂抗性突变体M9的遗传和基因克隆
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摘要
【目的】分离和鉴定自然突变的油菜乙酰乳酸合成酶抑制剂类除草剂抗性突变体M9抗性基因,阐明抗性产生的分子基础。【方法】通过配置杂交组合研究其遗传规律,应用同源序列法克隆油菜ALS家族基因,采用杂交转育和小孢子培养技术将抗性基因转育到陆奥-五十铃细胞质雄性不育(MICMS)恢复系中,进行PCR鉴定。【结果】该突变体抗性由1个显性核基因控制。克隆获得BnALS1—BnALS3,核酸序列比对发现,M9抗性是由BnALS1的点突变使蛋白序列的第638位丝氨酸(AGT)残基被天冬酰胺酸(AAT)替代所致,将此抗性基因命名为BnALS1R。抗性基因BnALS1R转育到MICMS恢复系中的PCR检测表明,所有抗除草剂的恢复系都携带有BnALS1R。【结论】突变体M9抗性由1个显性核基因控制,BnALS1第638位丝氨酸突变成天冬酰胺酸是抗性产生的分子基础。
【Objective】The objective of this study is to identify and characterize the resistant gene of a mutant line M9,which was previously found in the rapeseed(brassica napus L.) by spontaneous mutation,conferring resistance to acetolactate synthase or acetohydroxyacid synthase(ALS or AHAS) inhibiting herbicides,and understand the molecular basis for resistance to ALS-inhibiting herbicides.【Method】Reciprocal crosses were made in order to study the inheritance of resistance.Three genes BnALS1-BnALS3 encoding ALS were isolated from the mutant and wild type by using the homology-based candidate gene method.The resistant gene of M9 was transformed into Mutsu-Isuzu cytoplasmic male sterile(MICMS) restorer lines by hybridization,microspore culture,and PCR analysis.【Result】The resistance of M9 was inherited as a single,dominant nuclear gene.Three genes BnALS1-BnALS3 encoding ALS were isolated.Molecular analysis identified a single-point mutation leading to an amino acid substitution from serine 638(AGT) to asparagine(AAT) at the herbicide-binding site of the rapeseed BnALS1 gene.All the resistant restoring lines had the specific band of BnALS1R by sequencing.【Conclusion】The resistance trait of M9 is controlled by a single dominant nuclear gene.The molecular basis for the resistance to ALS-inhibiting herbicides results from the point mutation(Ser638Asn).
【Objective】The objective of this study is to identify and characterize the resistant gene of a mutant line M9,which was previously found in the rapeseed(brassica napus L.) by spontaneous mutation,conferring resistance to acetolactate synthase or acetohydroxyacid synthase(ALS or AHAS) inhibiting herbicides,and understand the molecular basis for resistance to ALS-inhibiting herbicides.【Method】Reciprocal crosses were made in order to study the inheritance of resistance.Three genes BnALS1-BnALS3 encoding ALS were isolated from the mutant and wild type by using the homology-based candidate gene method.The resistant gene of M9 was transformed into Mutsu-Isuzu cytoplasmic male sterile(MICMS) restorer lines by hybridization,microspore culture,and PCR analysis.【Result】The resistance of M9 was inherited as a single,dominant nuclear gene.Three genes BnALS1-BnALS3 encoding ALS were isolated.Molecular analysis identified a single-point mutation leading to an amino acid substitution from serine 638(AGT) to asparagine(AAT) at the herbicide-binding site of the rapeseed BnALS1 gene.All the resistant restoring lines had the specific band of BnALS1R by sequencing.【Conclusion】The resistance trait of M9 is controlled by a single dominant nuclear gene.The molecular basis for the resistance to ALS-inhibiting herbicides results from the point mutation(Ser638Asn).
引文
[1]Cai L,Zhou B W,Guo X L,Dong C H,Hu X J,Hou M S,Liu S Y.Pollen-mediated gene flow in Chinese commercial fields of glufosinate-resistant canola(Brassica napus).Chinese Science Bulletin,2008,53(15):2333-2341.
[2]Mary A R,Michael L,Christopher P,Stephen B P,Roush R T.Pollen-mediated movement of herbicide resistance between commercial canola fields.Science,2002,296:2386-2388.
[3]Stokstad E.A little pollen goes a long way.Science,2002,296:2314-2314.
[4]浦惠明,戚存扣,张洁夫,高建芹,傅寿仲,陈新军,陈松,赵祥祥.转基因抗除草剂油菜对近缘作物的基因漂移研究.生态学报,2005,25(3):196-203.Pu H M,Qi C K,Zhang J F,Gao J Q,Fu S Z,Chen X J,Chen S,Zhao X X.Studies on the gene flow from herbicide-tolerant GM rapeseed to its close relative crops.Acta Ecologica Sinica,2005,25(3):196-203.(in Chinese)
[5]浦惠明,戚存扣,张洁夫,高建芹,傅寿仲,陈新军,陈松,赵祥祥.转基因抗除草剂油菜对十字花科杂草的基因漂移研究.生态学报,2005,25(4):910-916.Pu H M,Qi C K,Zhang J F,Gao J Q,Fu S Z,Chen X J,Chen S,Zhao X X.The studies on gene flow from GM herbicide-tolerant rapeseed to cruciferous weeds.Acta Ecologica Sinica,2005,25(4):910-916.(inChinese)
[6]Chen H,Saksa K,Zhao F Y,Qiu J C,Xiong L M.Genetic analysis of pathway regulation for enhancing branched-chain amino acid biosynthesis in plants.The Plant Journal,2010,63:573-583.
[7]Ronald G D,Jennifer A M,Luke W G.Structure and mechanism of inhibition of plant acetohydroxyacid synthase.Plant Physiology Biochemistry,2008,46:309-324.
[8]Jennifer A M,Pang S S,Jack K S,Luke W G,Ronald G D.Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase.Proceedings of the National Academy of Sciences of the USA,2006,103:569-573.
[9]Tan S,Richard R E,Mark L D,Bijay K S,Dale L S.Imidazolinone-tolerant crops:History,current status and future.Pest Management Science,2005,61:246-257.
[10]Carlos A S,Mariano B,Mariel E,Sherry R,Ascenzi W R.Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower.Theoretical and Applied Genetics,2008,118:105-112.
[11]Rodriguez S C,Ramirez M C,Martinez C,Nadal S,Martin A,Atienza S G.Selection and molecular characterization of imidazolinone resistant mutation-derived lines of Tritordeum HT621.Molecular Breeding,2009,23:565-572.
[12]Pozniak C J,Birk I T,O’Donoughue L S,Christiane M,Pierre J H,Bijay K S.Physiological and molecular characterization of mutation-derived imidazolinone resistance in spring wheat.Crop Science,2004,44:1434-1443.
[13]Carlos A S,Mariano B.Inheritance and molecular characterization of broad range tolerance to herbicides targeting acetohydroxyacid synthase in sunflower.Theoretical and Applied Genetics,2012,124:355-364.
[14]Carlos A S,Mariano B,Emiliano A,Brigitte W.Response to imazapyr and dominance relationships of two imidazolinone-tolerant alleles at the Ahasl1locus of sunflower.Theoretical and Applied Genetics,2012,124:385-396.
[15]Hyejin L,Sachin R,Neeraj K,Burkea I,Yenisha J P,Gilla K S,Wettsteina D V,Steven E U.Single nucleotide mutation in the barley acetohydroxy acid synthase(AHAS)gene confers resistance to imidazolinone herbicides.Proceedings of the National Academy of Sciences of the USA,2011,108(21):8909-8913.
[16]Whaley C M,Wilson H P,Westwood J H.A new mutation in plant Als confers resistance to five classes of Als-inhibiting herbicides.Weed Science,2007,55(2):83-90.
[17]Swanson E B,Herrgesell M J,Arnoldo M,Sippell D W,Wong R S C.Microspore mutagenesis and selection:Canola plants with field tolerance to the imidazolinones.Theoretical and Applied Genetics,1989,78:525-530.
[18]Hattori J,Brown D,Mourad G,Labbe H,Ouellet T,Sunohara G,Rutledge R,King J,Miki B.An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance.Molecular and General Genetics,1995,246:419-425.
[19]高建芹,浦惠明,戚存扣,张洁夫,龙卫华,胡茂龙,陈松,陈新军,陈锋,顾慧.抗咪唑啉酮油菜种质的发现与鉴定.植物遗传资源学报,2010,11(3):369-373.Gao J Q,Pu H M,Qi C K,Zhang J F,Long W H,Hu M L,Chen S,Chen X J,Chen F,Gu H.Identification of imidazolidone-resistant oilseed rape mutant.Journal of Plant Genetic Resources,2010,11(3):369-373.(in Chinese)
[20]高建芹,浦惠明,龙卫华,胡茂龙,戚存扣,张洁夫.抗咪唑啉酮油菜对非选择性除草剂的抗性效应.江苏农业学报,2010,26(6):1186-1119.Gao J Q,Pu H M,Long W H,Hu M L,Qi C K,Zhang J F.Resistance of imidzolidone--resistant oilseed rape to nonselective Herbicide imazethapyr.Jiangsu Journal of Agriculture Science,2010,26(6):1186-1191.(in Chinese)
[21]胡茂龙,龙卫华,高建芹,陈新军,张洁夫,陈松,戚存扣,浦惠明.甘蓝型油菜谷胱甘肽过氧化物酶基因克隆和非生物胁迫下的表达.中国油料作物学报,2011,33(4):331-337.Hu M L,Long W H,Gao J Q,Chen X J,Zhang J F,Chen S,Qi C K,Pu H M.Cloning of glutathione peroxidase BnGPX1gene from Brassica napus L.and its expression under abiotic stresses.Chinese Journal of Oil Crop Sciences,2011,33(4):331-337.(in Chinese)
[22]龙卫华,浦惠明,戚存扣,张洁夫,陈松,陈新军,高建芹,陈锋,胡茂龙.油菜小孢子高校培养体系构建:Ⅰ影响小孢子成胚的若干因素研究.江苏农业学报,2008,24(6):792-796.Long W H,Pu H M,Qi C K,Zhang J F,Chen S,Chen X J,Gao J Q,Chen F,Hu M L.High efficiency microspore culture systemconstruction of Brassica napus:I.factors involved in microspore embryogenesis.Jiangsu Journal of Agriculture Science,2008,24(6):792-796.(in Chinese)
[23]Chen X,Truksa M,Snyder C L,El-Mezawy A,Shah S,Weselake R J.Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase4exhibit different expression patterns and functional divergence in Brassica napus.Plant Physiology,2011,155:851-865.
[24]Cheung F,Trick M,Drou N,Lim Y P,Park J Y,Kwon S J,Kim J A,Scott R,Pires J C,Paterson A H,Town C,Bancroft I.Comparative analysis between homoeologous genome segments of Brassica napus and its progenitor species reveals extensive sequence-level divergence.The Plant Cell,2009,21:1912-1928.
[25]Rutledge R G,Quellet T,Hattori J,Miki B L.Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family.Molecular and General Genetics,1991,229:31-40.
[26]Bekkaoui F,Janet A C,David A N,Maurice M M,William L C.Isolation,structure and expression of a cDNA for acetolactate synthase from Brassica napus.Plant Molecular Biology,1991,16:741-744.
[27]Ouellet T,Rutledget R G,Miki B L.Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression.The Plant Journal,1992,2:321-330.
[28]Yu Q,Han H P,Martin M,Vila-Aiub,Powles S B.AHAS herbicide resistance endowing mutations:Effect on AHAS functionality and plant growth.Journal of Experiment Botany,2010,61(14):3925-3934.
[29]Powles S B,Yu Q.Evolution in action:Plants resistant to herbicides.Annual Review of Plant Biology,2010,61:317-347.
[30]Stewart C N J.Weedy and Invasive Plant Genomics.Ed.Oxford:Blackwell,2009.
[2]Mary A R,Michael L,Christopher P,Stephen B P,Roush R T.Pollen-mediated movement of herbicide resistance between commercial canola fields.Science,2002,296:2386-2388.
[3]Stokstad E.A little pollen goes a long way.Science,2002,296:2314-2314.
[4]浦惠明,戚存扣,张洁夫,高建芹,傅寿仲,陈新军,陈松,赵祥祥.转基因抗除草剂油菜对近缘作物的基因漂移研究.生态学报,2005,25(3):196-203.Pu H M,Qi C K,Zhang J F,Gao J Q,Fu S Z,Chen X J,Chen S,Zhao X X.Studies on the gene flow from herbicide-tolerant GM rapeseed to its close relative crops.Acta Ecologica Sinica,2005,25(3):196-203.(in Chinese)
[5]浦惠明,戚存扣,张洁夫,高建芹,傅寿仲,陈新军,陈松,赵祥祥.转基因抗除草剂油菜对十字花科杂草的基因漂移研究.生态学报,2005,25(4):910-916.Pu H M,Qi C K,Zhang J F,Gao J Q,Fu S Z,Chen X J,Chen S,Zhao X X.The studies on gene flow from GM herbicide-tolerant rapeseed to cruciferous weeds.Acta Ecologica Sinica,2005,25(4):910-916.(inChinese)
[6]Chen H,Saksa K,Zhao F Y,Qiu J C,Xiong L M.Genetic analysis of pathway regulation for enhancing branched-chain amino acid biosynthesis in plants.The Plant Journal,2010,63:573-583.
[7]Ronald G D,Jennifer A M,Luke W G.Structure and mechanism of inhibition of plant acetohydroxyacid synthase.Plant Physiology Biochemistry,2008,46:309-324.
[8]Jennifer A M,Pang S S,Jack K S,Luke W G,Ronald G D.Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase.Proceedings of the National Academy of Sciences of the USA,2006,103:569-573.
[9]Tan S,Richard R E,Mark L D,Bijay K S,Dale L S.Imidazolinone-tolerant crops:History,current status and future.Pest Management Science,2005,61:246-257.
[10]Carlos A S,Mariano B,Mariel E,Sherry R,Ascenzi W R.Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower.Theoretical and Applied Genetics,2008,118:105-112.
[11]Rodriguez S C,Ramirez M C,Martinez C,Nadal S,Martin A,Atienza S G.Selection and molecular characterization of imidazolinone resistant mutation-derived lines of Tritordeum HT621.Molecular Breeding,2009,23:565-572.
[12]Pozniak C J,Birk I T,O’Donoughue L S,Christiane M,Pierre J H,Bijay K S.Physiological and molecular characterization of mutation-derived imidazolinone resistance in spring wheat.Crop Science,2004,44:1434-1443.
[13]Carlos A S,Mariano B.Inheritance and molecular characterization of broad range tolerance to herbicides targeting acetohydroxyacid synthase in sunflower.Theoretical and Applied Genetics,2012,124:355-364.
[14]Carlos A S,Mariano B,Emiliano A,Brigitte W.Response to imazapyr and dominance relationships of two imidazolinone-tolerant alleles at the Ahasl1locus of sunflower.Theoretical and Applied Genetics,2012,124:385-396.
[15]Hyejin L,Sachin R,Neeraj K,Burkea I,Yenisha J P,Gilla K S,Wettsteina D V,Steven E U.Single nucleotide mutation in the barley acetohydroxy acid synthase(AHAS)gene confers resistance to imidazolinone herbicides.Proceedings of the National Academy of Sciences of the USA,2011,108(21):8909-8913.
[16]Whaley C M,Wilson H P,Westwood J H.A new mutation in plant Als confers resistance to five classes of Als-inhibiting herbicides.Weed Science,2007,55(2):83-90.
[17]Swanson E B,Herrgesell M J,Arnoldo M,Sippell D W,Wong R S C.Microspore mutagenesis and selection:Canola plants with field tolerance to the imidazolinones.Theoretical and Applied Genetics,1989,78:525-530.
[18]Hattori J,Brown D,Mourad G,Labbe H,Ouellet T,Sunohara G,Rutledge R,King J,Miki B.An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance.Molecular and General Genetics,1995,246:419-425.
[19]高建芹,浦惠明,戚存扣,张洁夫,龙卫华,胡茂龙,陈松,陈新军,陈锋,顾慧.抗咪唑啉酮油菜种质的发现与鉴定.植物遗传资源学报,2010,11(3):369-373.Gao J Q,Pu H M,Qi C K,Zhang J F,Long W H,Hu M L,Chen S,Chen X J,Chen F,Gu H.Identification of imidazolidone-resistant oilseed rape mutant.Journal of Plant Genetic Resources,2010,11(3):369-373.(in Chinese)
[20]高建芹,浦惠明,龙卫华,胡茂龙,戚存扣,张洁夫.抗咪唑啉酮油菜对非选择性除草剂的抗性效应.江苏农业学报,2010,26(6):1186-1119.Gao J Q,Pu H M,Long W H,Hu M L,Qi C K,Zhang J F.Resistance of imidzolidone--resistant oilseed rape to nonselective Herbicide imazethapyr.Jiangsu Journal of Agriculture Science,2010,26(6):1186-1191.(in Chinese)
[21]胡茂龙,龙卫华,高建芹,陈新军,张洁夫,陈松,戚存扣,浦惠明.甘蓝型油菜谷胱甘肽过氧化物酶基因克隆和非生物胁迫下的表达.中国油料作物学报,2011,33(4):331-337.Hu M L,Long W H,Gao J Q,Chen X J,Zhang J F,Chen S,Qi C K,Pu H M.Cloning of glutathione peroxidase BnGPX1gene from Brassica napus L.and its expression under abiotic stresses.Chinese Journal of Oil Crop Sciences,2011,33(4):331-337.(in Chinese)
[22]龙卫华,浦惠明,戚存扣,张洁夫,陈松,陈新军,高建芹,陈锋,胡茂龙.油菜小孢子高校培养体系构建:Ⅰ影响小孢子成胚的若干因素研究.江苏农业学报,2008,24(6):792-796.Long W H,Pu H M,Qi C K,Zhang J F,Chen S,Chen X J,Gao J Q,Chen F,Hu M L.High efficiency microspore culture systemconstruction of Brassica napus:I.factors involved in microspore embryogenesis.Jiangsu Journal of Agriculture Science,2008,24(6):792-796.(in Chinese)
[23]Chen X,Truksa M,Snyder C L,El-Mezawy A,Shah S,Weselake R J.Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase4exhibit different expression patterns and functional divergence in Brassica napus.Plant Physiology,2011,155:851-865.
[24]Cheung F,Trick M,Drou N,Lim Y P,Park J Y,Kwon S J,Kim J A,Scott R,Pires J C,Paterson A H,Town C,Bancroft I.Comparative analysis between homoeologous genome segments of Brassica napus and its progenitor species reveals extensive sequence-level divergence.The Plant Cell,2009,21:1912-1928.
[25]Rutledge R G,Quellet T,Hattori J,Miki B L.Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family.Molecular and General Genetics,1991,229:31-40.
[26]Bekkaoui F,Janet A C,David A N,Maurice M M,William L C.Isolation,structure and expression of a cDNA for acetolactate synthase from Brassica napus.Plant Molecular Biology,1991,16:741-744.
[27]Ouellet T,Rutledget R G,Miki B L.Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression.The Plant Journal,1992,2:321-330.
[28]Yu Q,Han H P,Martin M,Vila-Aiub,Powles S B.AHAS herbicide resistance endowing mutations:Effect on AHAS functionality and plant growth.Journal of Experiment Botany,2010,61(14):3925-3934.
[29]Powles S B,Yu Q.Evolution in action:Plants resistant to herbicides.Annual Review of Plant Biology,2010,61:317-347.
[30]Stewart C N J.Weedy and Invasive Plant Genomics.Ed.Oxford:Blackwell,2009.