小麦B类MADS-box基因WPI1和WPI2的克隆及表达分析
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摘要
为探究小麦雌蕊化现象的分子遗传机制,本文从CSTP和HTS-1 2个小麦品系的幼穗中克隆得到了2个B类MADS-box基因WPI1和WPI2。WPI1基因c DNA序列长816 bp,ORF长627 bp,编码208个氨基酸残基;WPI2基因c DNA序列长983 bp,ORF长630 bp,编码209个氨基酸残基。WPI1和WPI2基因均含有典型的MADS-MEF2-like和K-box结构域,C端均具有PI结构基序。系统进化树分析表明WPI1和WPI2基因属于PI/GLO亚家族成员。Real-time PCR分析表明WPI1和WPI2基因在CSTP和HTS-1小穗中的表达模式明显不同。雌雄蕊原基形成期,WPI1和WPI2基因在HTS-1中的表达量较高,而在CSTP中表达量较低,接近为0;药隔期,WPI1和WPI2基因在HTS-1中的表达水平明显低于CSTP中的表达。WPI1和WPI2基因表达模式的改变与HTS-1雌蕊化表型有关。本研究结果为进一步探讨B类基因WPI1和WPI2在HTS-1雌蕊化中的作用奠定了基础。
To study the molecular and genetic mechanisms of pistillody in wheat,two B-class genes,WPI1 and WPI2 were cloned from CSTP and HTS-1 in this study. Sequence alignment results showed that the cloned c DNA of WPI1 and WPI2 was 816 bp and 983 bp in length,respectively. WPI1 gene contains a 627 bp open reading frame( ORF) which encodes 208 amino acids. WPI2 gene contains a 630 bp open reading frame( ORF) which encodes 209 amino acids.Alignment of the predicted amino acid sequences from WPI1 and WPI2 genes,as well as with that from previously published subfamily members,revealed the MADS-MEF2-like and K-box domains. In the two WPI genes,the consensus PI motif was found in the C-terminal region. Phylogenetic tree reconstructed clearly showed that these proteins were classed into PI / GLO subgroup of B class MADS-box protein family. Real-time PCR analysis indicated that the expression patterns of WPI1 and WPI2 genes are significantly different in spikelet of CSTP and HTS-1. In pistil and stamen primordiuxn formation stage the expression levels of WPI1 and WPI2 are higher in HTS-1 than CSTP,the latter is close to zero. In contrast,in anther seperation stage the expression levels of WPI1 and WPI2 are lower in HTS-1 than CSTP,obviously. WPI1 and WPI2 genes may be bound up with pistillody of HTS-1. The study provides more insights for future research the functions of B-class genes WPI1 and WPI2.
To study the molecular and genetic mechanisms of pistillody in wheat,two B-class genes,WPI1 and WPI2 were cloned from CSTP and HTS-1 in this study. Sequence alignment results showed that the cloned c DNA of WPI1 and WPI2 was 816 bp and 983 bp in length,respectively. WPI1 gene contains a 627 bp open reading frame( ORF) which encodes 208 amino acids. WPI2 gene contains a 630 bp open reading frame( ORF) which encodes 209 amino acids.Alignment of the predicted amino acid sequences from WPI1 and WPI2 genes,as well as with that from previously published subfamily members,revealed the MADS-MEF2-like and K-box domains. In the two WPI genes,the consensus PI motif was found in the C-terminal region. Phylogenetic tree reconstructed clearly showed that these proteins were classed into PI / GLO subgroup of B class MADS-box protein family. Real-time PCR analysis indicated that the expression patterns of WPI1 and WPI2 genes are significantly different in spikelet of CSTP and HTS-1. In pistil and stamen primordiuxn formation stage the expression levels of WPI1 and WPI2 are higher in HTS-1 than CSTP,the latter is close to zero. In contrast,in anther seperation stage the expression levels of WPI1 and WPI2 are lower in HTS-1 than CSTP,obviously. WPI1 and WPI2 genes may be bound up with pistillody of HTS-1. The study provides more insights for future research the functions of B-class genes WPI1 and WPI2.
引文
[1]高晶涵,李清昀.控制植物花器官发育的分子机理[J].生物学通报,2008,43(2):19-21
[2]Coen E S,Meyerowitz E M.The war of the whorls:genetic interactions controlling flower development[J].Nature,1991,353(5):31-37
[3]Skinner D J,Hill T A,Gasser C S.Regulation of ovule development[J].Plant Cell,2004,16(S1):32-45
[4]Angenent G C,Franken J,Busscher M,Dijken A,Went J L,Dons H J,Tunen A J.An novel class of MADS box genes is involved in ovule development in petunia[J].Plant Cell,1995,7(10):1569-1582
[5]Colombo L,Franken J,Koetje E,Went J L,Dons H J,Tunen A J.The Petunia MADS-box gene FBP11 determines ovule identity[J].Plant Cell,1995,7(11):1859-1868
[6]Benedito V A,Angenent G C,Tuyl J M,Krens F A.Lilium longiflorum and molecular floral development:The ABCDE model[J].Acta Horticulturae,2004,651(1):83-89
[7]Theiβen G,Saedler H.Plant biology:floral quartets[J].Nature,2001,409(6819):469-471
[8]Soltis D E,Ma H,Frohlich M W,Soltis P S,Albert V A,Oppenheimer D G,Altman N S,Pamphilis C,Leebens-Mack J.The floral genome:an evolutionary history of gene duplication and shifting patterns of gene expression[J].Trends in Plant Science,2007,12(8):358-367
[9]甘德芳,丁飞,庄丹,梁丹迪.黄瓜全基因组转录因子MADSbox家族基因的序列特征分析[J].核农学报,2012,26(9):1249-1256
[10]Münster T,Pahnke J,Di Rosa A,Kim J T,Martin W,Saedler H,Theissen G.Floral homeotic genes were recruited from homologous MADS-box genes preexisting in the common ancestor of ferns and seed plants[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(6):2415-2420
[11]Nam J,Kim J,Lee S,An J,Ma H,Nei M.TypeⅠMADS-box genes have experienced faster birth-and-death evolution than typeⅡMADS-box genes in angiosperms[J].Proceedings of the National Academy of Sciences of the United States of America,2003,101(7):1910-1915
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[13]Alvarez-Buylla E R,Pelaz S,Liljegren S J,Gold S E,Burgeff C,Ditta G S,De Pouplana L R,Martínez-Castilla L,Yanofsky M F.An ancestral MADS-box gene duplication occurred before the divergence of plants and animals[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(10):5328-5333
[14]Ng M,Yanofsky M F.Function and evolution of the plant MADSbox gene family[J].Nature Reviews Genetics,2001,2(3):186-195
[15]任磊,王雁,周琳,彭镇华.Ps MADS基因的克隆、表达及载体构建[J].核农学报,2011,25(5):939-944
[16]Becker A,Kaufmann K,Freialdenhoven A,Vincent C,Li MA,Saedler H,Theissen G.A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes[J].Molecular Genetics and Genomics,2002,266(6):942-950
[17]Hama E,Takumi S,Ogihara Y,Murai K.Pistillody is caused by alterations to the class-B MADS-box gene expression pattern in alloplasmic wheats[J].Planta,2004,218(5):712-720
[18]Kang H G,Jeon J S,Lee S,An G.Identication of class B and class Coral organ identity genes from rice plants[J].Plant Molecular Biology,1998,38(6):1021-1029
[19]杨在君,彭正松,周永红,彭丽娟,魏淑红.利用SRAP分子标记评价小麦三雌蕊近等基因系的遗传背景[J].核农学报,2012,26(1):22-27
[20]Peng Z S.A new mutation in wheat producing three pistils in a floret[J].Journal of Agronomy and Crop Science,2003,189(4):270-272
[21]Peng Z S,Yang J,Wei S H,Zeng J H.Characterization of common wheat(Triticum aestivum L.)mutation line producing three pistils in a floret[J].Hereditas,2004,141(1):15-18
[22]Murai K,Takumi S,Koga H,Ogihara Y.Pistillody,homeotic transformation of stamens into pistil-like structures,caused by nuclear-cytoplasm interaction in wheat[J].The Plant Journal,2002,29(2):169-181
[23]Murai K,Miyamae M,Kato H,Takumi S,Ogihara Y.WAP1,a wheat APETALA1 homolog,plays a central role in the phase transition from vegetative to reproductive growth[J].Plant Cell Physiology,2003,44(12):1255-1265
[24]王俊英,赵春江,杨宝柱.小麦小花发育与退化的研究[J].华北农学报,1996,11(2):9-13
[25]Yamada K,Saraike T,Shitsukawa N,Hirabayashi C,Takumi S,Murai K.Class D and B(sister)MADS-box genes are associated with ectopic ovule formation in the pistil-like stamens of alloplasmic wheat(Triticum aestivum L.)[J].Plant Molecular Biology,2009,71(1/2):1-14
[26]杨在君.小麦三雌蕊突变体幼穗中基因的差异表达分析[D].雅安:四川农业大学,2011:39-40
[27]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408
[28]Kramer E M,Dorit R L,Irish V F.Molecular evolution of genes controlling petal and stamen development:duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages[J].Genetics,1998,149(2):765-783
[29]Theiβen G.Development of floral organ identity:stories from the MADS house[J].Current Opinion in Plant Biology,2001,4(1):75-85
[30]Ferrario S,Immink R G H,Angenent G C.Conservation and diversity in flower land[J].Current Opinion in Plant Biology,2004,7(1):84-91
[31]Jack T,Brockman L L,Meyerowitz E M.The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS-box and is expressed in petals and stamens[J].Cell,1992,68(4):683-697
[32]Goto K,Meyerowitz E M.Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA[J].Genes and Development,1994,8(13):1548-1560
[33]Pelaz S,Tapia-López R,Alvarez-Buylla E R,Yanofsky MF.Conversion of leaves into petals in Arabidopsis[J].Current Biology,2001,11(3):182-184
[34]Hong-Guy K,Noh Y S,Chung Y Y,Costa M A,An K,An G.Phenotypic alterations of petal and sepal by ectopic expression of a rice MADS box gene in tobacco[J].Plant Molecular Biology,1995,29(1):1-10
[35]Honma T,Goto K.Complexes of MADS-box proteins are sufficient to convert leaves into floral organs[J].Nature,2001,409(6819):525-529
[36]Yoshida H,Itoh J,Ohmori S,Miyoshi K,Horigome A,Uchida E,Kimizu M,Matsumura Y,Kusaba M,Satoh H,Nagato Y.Superwoman-cleistogamy,a hopeful allele for gene containent in GM rice[J].Plant Biotechnology Journal,2007,5(6):835-846
[37]Meguro A,Takumi S,Ogihara Y,Murai K.WAG,a wheat AGAMOUS homolog,is associated with development of pistil-like stamens in alloplasmic wheats[J].Sexual Plant Reproduction,2003,15(5):221-230
[38]欧阳钟鸣.小麦雄蕊同源转化型不育新材料的遗传分析及不育基因的初步定位[D].南充:西华师范大学,2012:22-23
[2]Coen E S,Meyerowitz E M.The war of the whorls:genetic interactions controlling flower development[J].Nature,1991,353(5):31-37
[3]Skinner D J,Hill T A,Gasser C S.Regulation of ovule development[J].Plant Cell,2004,16(S1):32-45
[4]Angenent G C,Franken J,Busscher M,Dijken A,Went J L,Dons H J,Tunen A J.An novel class of MADS box genes is involved in ovule development in petunia[J].Plant Cell,1995,7(10):1569-1582
[5]Colombo L,Franken J,Koetje E,Went J L,Dons H J,Tunen A J.The Petunia MADS-box gene FBP11 determines ovule identity[J].Plant Cell,1995,7(11):1859-1868
[6]Benedito V A,Angenent G C,Tuyl J M,Krens F A.Lilium longiflorum and molecular floral development:The ABCDE model[J].Acta Horticulturae,2004,651(1):83-89
[7]Theiβen G,Saedler H.Plant biology:floral quartets[J].Nature,2001,409(6819):469-471
[8]Soltis D E,Ma H,Frohlich M W,Soltis P S,Albert V A,Oppenheimer D G,Altman N S,Pamphilis C,Leebens-Mack J.The floral genome:an evolutionary history of gene duplication and shifting patterns of gene expression[J].Trends in Plant Science,2007,12(8):358-367
[9]甘德芳,丁飞,庄丹,梁丹迪.黄瓜全基因组转录因子MADSbox家族基因的序列特征分析[J].核农学报,2012,26(9):1249-1256
[10]Münster T,Pahnke J,Di Rosa A,Kim J T,Martin W,Saedler H,Theissen G.Floral homeotic genes were recruited from homologous MADS-box genes preexisting in the common ancestor of ferns and seed plants[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(6):2415-2420
[11]Nam J,Kim J,Lee S,An J,Ma H,Nei M.TypeⅠMADS-box genes have experienced faster birth-and-death evolution than typeⅡMADS-box genes in angiosperms[J].Proceedings of the National Academy of Sciences of the United States of America,2003,101(7):1910-1915
[12]Alvarez-Buylla E R,Liljergren S J,Pelaz S,Gold S E,Burgeff C,Ditta G S,Vergara-Silva F,Yanofsky M F.MADS-box gene evolution beyond flowers:expression in pollen,endosperm,guard cells,roots and trichomes[J].The Plant Journal,2000,24(4):457-466
[13]Alvarez-Buylla E R,Pelaz S,Liljegren S J,Gold S E,Burgeff C,Ditta G S,De Pouplana L R,Martínez-Castilla L,Yanofsky M F.An ancestral MADS-box gene duplication occurred before the divergence of plants and animals[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(10):5328-5333
[14]Ng M,Yanofsky M F.Function and evolution of the plant MADSbox gene family[J].Nature Reviews Genetics,2001,2(3):186-195
[15]任磊,王雁,周琳,彭镇华.Ps MADS基因的克隆、表达及载体构建[J].核农学报,2011,25(5):939-944
[16]Becker A,Kaufmann K,Freialdenhoven A,Vincent C,Li MA,Saedler H,Theissen G.A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes[J].Molecular Genetics and Genomics,2002,266(6):942-950
[17]Hama E,Takumi S,Ogihara Y,Murai K.Pistillody is caused by alterations to the class-B MADS-box gene expression pattern in alloplasmic wheats[J].Planta,2004,218(5):712-720
[18]Kang H G,Jeon J S,Lee S,An G.Identication of class B and class Coral organ identity genes from rice plants[J].Plant Molecular Biology,1998,38(6):1021-1029
[19]杨在君,彭正松,周永红,彭丽娟,魏淑红.利用SRAP分子标记评价小麦三雌蕊近等基因系的遗传背景[J].核农学报,2012,26(1):22-27
[20]Peng Z S.A new mutation in wheat producing three pistils in a floret[J].Journal of Agronomy and Crop Science,2003,189(4):270-272
[21]Peng Z S,Yang J,Wei S H,Zeng J H.Characterization of common wheat(Triticum aestivum L.)mutation line producing three pistils in a floret[J].Hereditas,2004,141(1):15-18
[22]Murai K,Takumi S,Koga H,Ogihara Y.Pistillody,homeotic transformation of stamens into pistil-like structures,caused by nuclear-cytoplasm interaction in wheat[J].The Plant Journal,2002,29(2):169-181
[23]Murai K,Miyamae M,Kato H,Takumi S,Ogihara Y.WAP1,a wheat APETALA1 homolog,plays a central role in the phase transition from vegetative to reproductive growth[J].Plant Cell Physiology,2003,44(12):1255-1265
[24]王俊英,赵春江,杨宝柱.小麦小花发育与退化的研究[J].华北农学报,1996,11(2):9-13
[25]Yamada K,Saraike T,Shitsukawa N,Hirabayashi C,Takumi S,Murai K.Class D and B(sister)MADS-box genes are associated with ectopic ovule formation in the pistil-like stamens of alloplasmic wheat(Triticum aestivum L.)[J].Plant Molecular Biology,2009,71(1/2):1-14
[26]杨在君.小麦三雌蕊突变体幼穗中基因的差异表达分析[D].雅安:四川农业大学,2011:39-40
[27]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408
[28]Kramer E M,Dorit R L,Irish V F.Molecular evolution of genes controlling petal and stamen development:duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages[J].Genetics,1998,149(2):765-783
[29]Theiβen G.Development of floral organ identity:stories from the MADS house[J].Current Opinion in Plant Biology,2001,4(1):75-85
[30]Ferrario S,Immink R G H,Angenent G C.Conservation and diversity in flower land[J].Current Opinion in Plant Biology,2004,7(1):84-91
[31]Jack T,Brockman L L,Meyerowitz E M.The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS-box and is expressed in petals and stamens[J].Cell,1992,68(4):683-697
[32]Goto K,Meyerowitz E M.Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA[J].Genes and Development,1994,8(13):1548-1560
[33]Pelaz S,Tapia-López R,Alvarez-Buylla E R,Yanofsky MF.Conversion of leaves into petals in Arabidopsis[J].Current Biology,2001,11(3):182-184
[34]Hong-Guy K,Noh Y S,Chung Y Y,Costa M A,An K,An G.Phenotypic alterations of petal and sepal by ectopic expression of a rice MADS box gene in tobacco[J].Plant Molecular Biology,1995,29(1):1-10
[35]Honma T,Goto K.Complexes of MADS-box proteins are sufficient to convert leaves into floral organs[J].Nature,2001,409(6819):525-529
[36]Yoshida H,Itoh J,Ohmori S,Miyoshi K,Horigome A,Uchida E,Kimizu M,Matsumura Y,Kusaba M,Satoh H,Nagato Y.Superwoman-cleistogamy,a hopeful allele for gene containent in GM rice[J].Plant Biotechnology Journal,2007,5(6):835-846
[37]Meguro A,Takumi S,Ogihara Y,Murai K.WAG,a wheat AGAMOUS homolog,is associated with development of pistil-like stamens in alloplasmic wheats[J].Sexual Plant Reproduction,2003,15(5):221-230
[38]欧阳钟鸣.小麦雄蕊同源转化型不育新材料的遗传分析及不育基因的初步定位[D].南充:西华师范大学,2012:22-23