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        小麥GDH1基因克隆及其功能標(biāo)記開發(fā)

        2014-11-22 11:04:29李冰張照貴王佳佳
        山東農(nóng)業(yè)科學(xué) 2014年10期

        李冰 張照貴 王佳佳 等

        摘要:谷氨酸脫氫酶(glutamate dehydrogenase, GDH)在植物體內(nèi)催化合成谷氨酸的可逆反應(yīng),通過GDH固氮比谷氨酸合成酶途徑更節(jié)省能量。在植物大多數(shù)組織中,GDH1是該基因家族中表達(dá)最高的基因,比其他GDH成員具有更為重要的作用。本研究從普通小麥基因組中分離了TaGDH1基因在A、B、D染色體組的序列。針對(duì)TaGDH1a基因在基因組DNA序列1 900~1 983 bp位置存在的核苷酸差異設(shè)計(jì)了一個(gè)插入/缺失標(biāo)記,同時(shí)將該標(biāo)記定位在5A染色體上。

        關(guān)鍵詞:小麥;TaGDH1;同源克?。还δ軜?biāo)記

        中圖分類號(hào):S512.103.3文獻(xiàn)標(biāo)識(shí)號(hào):A文章編號(hào):1001-4942(2014)10-0006-06

        3討論

        普通小麥為異源六倍體(2n=6x=42),具有六個(gè)染色體組,在二倍體物種中為單拷貝的基因在普通小麥中可能有3個(gè)拷貝,分別由A、B和D三個(gè)亞基因組編碼,因此區(qū)分基因所在的染色體組比較困難[20]。郝麗芳等[21]在對(duì)小麥NOA基因所屬染色體組區(qū)分時(shí),設(shè)計(jì)兩對(duì)保守的跨內(nèi)含子的引物,通過基因組PCR、克隆和測(cè)序后的序列多態(tài)性分析,確定了小麥基因組中至少存在3個(gè)NOA成員,結(jié)合基于毛細(xì)管電泳的片段分析將3個(gè)成員定位在6A、6B和6D染色體上。李亞青[22]等以中國(guó)春缺體-四體系為材料,用Southern雜交的方法將TaGSK1基因定位于第一同源群的1A、1B和1D染色體。張磊等[23]利用特異引物在中國(guó)春缺體-四體中擴(kuò)增產(chǎn)物的長(zhǎng)度差異,將TaCKX5基因定位在小麥的3A、3B和3D染色體上。本研究利用二倍體、四倍體中各染色體組與六倍體小麥中的相對(duì)應(yīng)染色體組基因相似度高的特點(diǎn),將通過基因克隆獲得的二倍體、四倍體GDH1基因與六倍體小麥中的TaGDH1基因序列進(jìn)行對(duì)比,以區(qū)分三條TaGDH1基因所屬的染色體組。同時(shí)結(jié)合中國(guó)春缺體-四體染色體定位和RIL群體連鎖分析的方法將TaGDH1a-InDel標(biāo)記定位在5A染色體上。

        Andersen等[24]首先提出基因功能標(biāo)記概念,功能標(biāo)記的多態(tài)性來源于造成等位基因功能差異的DNA序列差異,可以進(jìn)行基因型鑒定和基因型選擇。因此,開發(fā)功能標(biāo)記對(duì)于提高小麥育種效率具有重要意義。本研究中TaGDH1a基因功能標(biāo)記的開發(fā)有助于GDH1在小麥中功能的研究以及TaGDH1基因型的鑒別。碳、氮代謝直接影響作物經(jīng)濟(jì)產(chǎn)量。已有研究表明,小麥中大部分與馴化和產(chǎn)量形成有關(guān)的QTL位點(diǎn)都聚集在第一和第五同源群染色體上[25, 26]。其中5A染色體上已發(fā)現(xiàn)存在控制穗長(zhǎng)、穗粒數(shù)、穗粒重等小麥產(chǎn)量性狀的主效QTL位點(diǎn)區(qū)域[27, 28]。本研究通過中國(guó)春缺體-四體系統(tǒng),結(jié)合連鎖分析的方法,成功將TaGDH1a基因定位到了小麥5A染色體上,為今后結(jié)合5A上已知產(chǎn)量性狀相關(guān)QTL研究TaGDH1基因功能奠定了基礎(chǔ)。

        參考文獻(xiàn):

        [1]

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        [8]馬敬坤, 袁永澤, 歐吉權(quán), 等. 外源水楊酸對(duì)水稻(Oryza sativa L.) 幼苗根的 NaCl 脅迫緩解效應(yīng)[J]. 武漢大學(xué)學(xué)報(bào): 理學(xué)版, 2006, 52(4):471-474.

        [9]Lu B, Yuan Y, Zhang C, et al. Modulation of key enzymes involved in ammonium assimilation and carbon metabolism by low temperature in rice (Oryza sativa L.) roots [J]. Plant Science, 2005, 169:295-302.

        [10]Qiu X, Xie W, Lian X, et al. Molecular analyses of the rice glutamate dehydrogenase gene family and their response to nitrogen and phosphorous deprivation [J]. Plant Cell Reports, 2009, 28: 1115-1126.

        [11]Tercé-Laforgue T, Bedu M, Dargel-Grafin C, et al. Resolving the role of plant glutamate dehydrogenase: II. physiological characterization of plants overexpressing the two enzyme subunits individually or simultaneously [J]. Plant and Cell Physiology, 2013, 54: 1635-1647.

        [12]Hirel B, Bertin P, Quilleré I, et al. Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize [J]. Plant Physiology, 2001, 125: 1258-1270.

        [13]Limami A M, Rouillon C, Glevarec G, et al. Genetic and physiological analysis of germination efficiency in maize in relation to nitrogen metabolism reveals the importance of cytosolic glutamine synthetase [J]. Plant Physiology, 2002, 130: 1860-1870.

        [14]Obara M, Kajiura M, Fukuta Y, et al. Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice (Oryza sativa L.) [J]. Journal of Experimental Botany, 2001, 52: 1209-1217.

        [15]Bagge M, Xia X, Lübberstedt T. Functional markers in wheat [J]. Current Opinion in Plant Biology, 2007, 10: 211-216.

        [16]喬麟軼, 張磊, 張文萍, 等. 小麥生長(zhǎng)素結(jié)合基因TaABP1-D的克隆、功能標(biāo)記開發(fā)及其與株高的關(guān)聯(lián)[J]. 作物學(xué)報(bào), 2012, 38(11): 2034-2041.

        [17]劉亞男. 普通小麥細(xì)胞壁轉(zhuǎn)化酶基因TaCwi-Al的表達(dá)和直立密穗(DEP1)基因克隆與功能標(biāo)記開發(fā)[D].北京:中國(guó)農(nóng)業(yè)科學(xué)院, 2012.

        [18]Devos K M, Gale M D. The use of random amplified polymorphic DNA markers in wheat [J]. Theoretical and Applied Genetics, 1992, 84: 567-572.

        [19]Haudry A, Cenci A, Ravel C, et al. Grinding up wheat: a massive loss of nucleotide diversity since domestication[J]. Mol. Biol. Evol., 2007, 24: 1506-1517.

        [20]Lazo G R, Chao S, Hummel D D, et al. Development of an expressed sequence tag (EST) resource for wheat (Triticum aestivum L.) EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bin-delineated map [J]. Genetics, 2004, 168: 585-593.

        [21]郝麗芳, 余春梅, 李斌, 等. 普通小麥中一氧化氮相關(guān)因子 (TaNOA) 編碼基因的克隆和分子生物學(xué)分析[J]. 生物工程學(xué)報(bào), 2010, 26: 48-56.

        [22]李亞青,毛新國(guó),趙寶存,等. 小麥糖原合成酶激酶基因(TaGSK1)的染色體定位[J]. 華北農(nóng)學(xué)報(bào),2006,21(5):39-41.

        [23]張磊, 張寶石, 周榮華, 等. 小麥細(xì)胞分裂素氧化/脫氫酶基因(TaCKX5)的克隆及其染色體定位[J]. 中國(guó)農(nóng)業(yè)科學(xué), 2008, 41(3):636-642.

        [24]Andersen J R, Lbberstedt T. Functional marks in plants [J]. Trends in Plant Science, 2003, 8:554-560.

        [25]Peng J, Ronin Y, Fahima T, et al. Domestication quantitative trait loci in Triticum dicoccoides, the progenitor of wheat [J]. Proceedings of the National Academy of Sciences, 2003, 100: 2489-2494.

        [26]Brner A, Schumann E, Fürste A, et al. Mapping of quantitative trait loci determining agronomic important characters in hexaploid wheat (Triticum aestivum L.) [J]. Theoretical and Applied Genetics, 2002, 105: 921-936.

        [27]Jantasuriyarat C, Vales M I, Watson C J W, et al. Identification and mapping of genetic loci affecting the free-threshing habit and spike compactness in wheat (Triticum aestivum L.) [J]. Theoretical and Applied Genetics, 2004, 108: 261-273.

        [28]Kato K, Miura H, Sawada S. Mapping QTLs controlling grain yield and its components on chromosome 5A of wheat [J]. Theoretical and Applied Genetics, 2000, 101: 1114-1121.

        [9]Lu B, Yuan Y, Zhang C, et al. Modulation of key enzymes involved in ammonium assimilation and carbon metabolism by low temperature in rice (Oryza sativa L.) roots [J]. Plant Science, 2005, 169:295-302.

        [10]Qiu X, Xie W, Lian X, et al. Molecular analyses of the rice glutamate dehydrogenase gene family and their response to nitrogen and phosphorous deprivation [J]. Plant Cell Reports, 2009, 28: 1115-1126.

        [11]Tercé-Laforgue T, Bedu M, Dargel-Grafin C, et al. Resolving the role of plant glutamate dehydrogenase: II. physiological characterization of plants overexpressing the two enzyme subunits individually or simultaneously [J]. Plant and Cell Physiology, 2013, 54: 1635-1647.

        [12]Hirel B, Bertin P, Quilleré I, et al. Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize [J]. Plant Physiology, 2001, 125: 1258-1270.

        [13]Limami A M, Rouillon C, Glevarec G, et al. Genetic and physiological analysis of germination efficiency in maize in relation to nitrogen metabolism reveals the importance of cytosolic glutamine synthetase [J]. Plant Physiology, 2002, 130: 1860-1870.

        [14]Obara M, Kajiura M, Fukuta Y, et al. Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice (Oryza sativa L.) [J]. Journal of Experimental Botany, 2001, 52: 1209-1217.

        [15]Bagge M, Xia X, Lübberstedt T. Functional markers in wheat [J]. Current Opinion in Plant Biology, 2007, 10: 211-216.

        [16]喬麟軼, 張磊, 張文萍, 等. 小麥生長(zhǎng)素結(jié)合基因TaABP1-D的克隆、功能標(biāo)記開發(fā)及其與株高的關(guān)聯(lián)[J]. 作物學(xué)報(bào), 2012, 38(11): 2034-2041.

        [17]劉亞男. 普通小麥細(xì)胞壁轉(zhuǎn)化酶基因TaCwi-Al的表達(dá)和直立密穗(DEP1)基因克隆與功能標(biāo)記開發(fā)[D].北京:中國(guó)農(nóng)業(yè)科學(xué)院, 2012.

        [18]Devos K M, Gale M D. The use of random amplified polymorphic DNA markers in wheat [J]. Theoretical and Applied Genetics, 1992, 84: 567-572.

        [19]Haudry A, Cenci A, Ravel C, et al. Grinding up wheat: a massive loss of nucleotide diversity since domestication[J]. Mol. Biol. Evol., 2007, 24: 1506-1517.

        [20]Lazo G R, Chao S, Hummel D D, et al. Development of an expressed sequence tag (EST) resource for wheat (Triticum aestivum L.) EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bin-delineated map [J]. Genetics, 2004, 168: 585-593.

        [21]郝麗芳, 余春梅, 李斌, 等. 普通小麥中一氧化氮相關(guān)因子 (TaNOA) 編碼基因的克隆和分子生物學(xué)分析[J]. 生物工程學(xué)報(bào), 2010, 26: 48-56.

        [22]李亞青,毛新國(guó),趙寶存,等. 小麥糖原合成酶激酶基因(TaGSK1)的染色體定位[J]. 華北農(nóng)學(xué)報(bào),2006,21(5):39-41.

        [23]張磊, 張寶石, 周榮華, 等. 小麥細(xì)胞分裂素氧化/脫氫酶基因(TaCKX5)的克隆及其染色體定位[J]. 中國(guó)農(nóng)業(yè)科學(xué), 2008, 41(3):636-642.

        [24]Andersen J R, Lbberstedt T. Functional marks in plants [J]. Trends in Plant Science, 2003, 8:554-560.

        [25]Peng J, Ronin Y, Fahima T, et al. Domestication quantitative trait loci in Triticum dicoccoides, the progenitor of wheat [J]. Proceedings of the National Academy of Sciences, 2003, 100: 2489-2494.

        [26]Brner A, Schumann E, Fürste A, et al. Mapping of quantitative trait loci determining agronomic important characters in hexaploid wheat (Triticum aestivum L.) [J]. Theoretical and Applied Genetics, 2002, 105: 921-936.

        [27]Jantasuriyarat C, Vales M I, Watson C J W, et al. Identification and mapping of genetic loci affecting the free-threshing habit and spike compactness in wheat (Triticum aestivum L.) [J]. Theoretical and Applied Genetics, 2004, 108: 261-273.

        [28]Kato K, Miura H, Sawada S. Mapping QTLs controlling grain yield and its components on chromosome 5A of wheat [J]. Theoretical and Applied Genetics, 2000, 101: 1114-1121.

        [9]Lu B, Yuan Y, Zhang C, et al. Modulation of key enzymes involved in ammonium assimilation and carbon metabolism by low temperature in rice (Oryza sativa L.) roots [J]. Plant Science, 2005, 169:295-302.

        [10]Qiu X, Xie W, Lian X, et al. Molecular analyses of the rice glutamate dehydrogenase gene family and their response to nitrogen and phosphorous deprivation [J]. Plant Cell Reports, 2009, 28: 1115-1126.

        [11]Tercé-Laforgue T, Bedu M, Dargel-Grafin C, et al. Resolving the role of plant glutamate dehydrogenase: II. physiological characterization of plants overexpressing the two enzyme subunits individually or simultaneously [J]. Plant and Cell Physiology, 2013, 54: 1635-1647.

        [12]Hirel B, Bertin P, Quilleré I, et al. Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize [J]. Plant Physiology, 2001, 125: 1258-1270.

        [13]Limami A M, Rouillon C, Glevarec G, et al. Genetic and physiological analysis of germination efficiency in maize in relation to nitrogen metabolism reveals the importance of cytosolic glutamine synthetase [J]. Plant Physiology, 2002, 130: 1860-1870.

        [14]Obara M, Kajiura M, Fukuta Y, et al. Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice (Oryza sativa L.) [J]. Journal of Experimental Botany, 2001, 52: 1209-1217.

        [15]Bagge M, Xia X, Lübberstedt T. Functional markers in wheat [J]. Current Opinion in Plant Biology, 2007, 10: 211-216.

        [16]喬麟軼, 張磊, 張文萍, 等. 小麥生長(zhǎng)素結(jié)合基因TaABP1-D的克隆、功能標(biāo)記開發(fā)及其與株高的關(guān)聯(lián)[J]. 作物學(xué)報(bào), 2012, 38(11): 2034-2041.

        [17]劉亞男. 普通小麥細(xì)胞壁轉(zhuǎn)化酶基因TaCwi-Al的表達(dá)和直立密穗(DEP1)基因克隆與功能標(biāo)記開發(fā)[D].北京:中國(guó)農(nóng)業(yè)科學(xué)院, 2012.

        [18]Devos K M, Gale M D. The use of random amplified polymorphic DNA markers in wheat [J]. Theoretical and Applied Genetics, 1992, 84: 567-572.

        [19]Haudry A, Cenci A, Ravel C, et al. Grinding up wheat: a massive loss of nucleotide diversity since domestication[J]. Mol. Biol. Evol., 2007, 24: 1506-1517.

        [20]Lazo G R, Chao S, Hummel D D, et al. Development of an expressed sequence tag (EST) resource for wheat (Triticum aestivum L.) EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bin-delineated map [J]. Genetics, 2004, 168: 585-593.

        [21]郝麗芳, 余春梅, 李斌, 等. 普通小麥中一氧化氮相關(guān)因子 (TaNOA) 編碼基因的克隆和分子生物學(xué)分析[J]. 生物工程學(xué)報(bào), 2010, 26: 48-56.

        [22]李亞青,毛新國(guó),趙寶存,等. 小麥糖原合成酶激酶基因(TaGSK1)的染色體定位[J]. 華北農(nóng)學(xué)報(bào),2006,21(5):39-41.

        [23]張磊, 張寶石, 周榮華, 等. 小麥細(xì)胞分裂素氧化/脫氫酶基因(TaCKX5)的克隆及其染色體定位[J]. 中國(guó)農(nóng)業(yè)科學(xué), 2008, 41(3):636-642.

        [24]Andersen J R, Lbberstedt T. Functional marks in plants [J]. Trends in Plant Science, 2003, 8:554-560.

        [25]Peng J, Ronin Y, Fahima T, et al. Domestication quantitative trait loci in Triticum dicoccoides, the progenitor of wheat [J]. Proceedings of the National Academy of Sciences, 2003, 100: 2489-2494.

        [26]Brner A, Schumann E, Fürste A, et al. Mapping of quantitative trait loci determining agronomic important characters in hexaploid wheat (Triticum aestivum L.) [J]. Theoretical and Applied Genetics, 2002, 105: 921-936.

        [27]Jantasuriyarat C, Vales M I, Watson C J W, et al. Identification and mapping of genetic loci affecting the free-threshing habit and spike compactness in wheat (Triticum aestivum L.) [J]. Theoretical and Applied Genetics, 2004, 108: 261-273.

        [28]Kato K, Miura H, Sawada S. Mapping QTLs controlling grain yield and its components on chromosome 5A of wheat [J]. Theoretical and Applied Genetics, 2000, 101: 1114-1121.

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