K、V、T型小麦细胞质雄性不育系叶绿体DNA的SSR分析及RuBP羧化酶活性比较
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摘要
为了探讨小麦细胞质雄性不育(CMS)与叶绿体DNA(cpDNA)的关系,揭示CMS机理。以2套K、V、T型同核异质不育系(A)及其保持系(B)‘太911289’和‘冀5418’、育性恢复的F_1和各自的质供体粘果山羊草(Aegilops kotschyi)、偏凸山羊草(Aegilops ventricosa)、提莫菲维小麦(Triticum timopheevii)为试验材料,利用cpSSR引物对小麦cpDNA进行比较分析,筛选出代表不同胞质不育系的引物,探讨CMS与叶绿体的关系;同时测定由叶绿体DNA与核DNA共同编码的RuBP羧化酶的活性,为小麦K、V、T雄性不育类型的应用提供理论依据。结果表明:(1)K型、V型、T型不育系与保持系的cpDNA之间均呈现多态性,且不同的细胞质之间存在特异片段,这从DNA水平上提供了3类不育系胞质来源不同的证据,并分别找到5对和7对可以鉴定V型和T型不育系的特异引物。(2)除K型‘冀5418’与其胞质供体的cpDNA出现差异外,不育系与其胞质供体的cpDNA没有差异,而且不育系与其育性恢复的F_1代cpSSR扩增片段之间也没有差异,很好地遗传了其母本性状。(3)在起身期和开花期,不仅2套不育系的RuBP羧化酶活性显著高于保持系,且在不育系与恢复系杂交的F_1中,随着育性的恢复其RuBP羧化酶活性高于保持系而低于各自不育系;而在拔节期的不育系与保持系之间、不育系之间、以及F_1代与不育系和保持系之间的RuBP酶活性大小没有显著差异。这可能与拔节期主要是营养生长有关系。
Cytoplasmic male-sterile(CMS)lines,especially K(Aegilops kotchyi),V(Aegilops ventricosa),and T(Triticum timopheevii)types of cytoplasm,are of high value in heterosis utilization in wheat(Triticum aestivum L.).The aim of this study was to discuss the relationship between CMS and chloroplast and to reveal the mechanism of CMS by investigating chloroplast DNA and RuBP carboxylase(rubi-sco)activity in CMS male-sterile lines.The experiment was conducted with cultivars of the K,V,T-type CMS lines,the common maintainer‘Tai 911289'and‘Ji 5418',the F_1 hybrids and the cytoplasmic donors under field conditions.The major results are as follows:(1)the polymorphism between male sterile lines and maintainer lines in cpDNA,and specific fragment in the different cytoplasm type male sterile lines provides evidence of three types of cytoplasm male sterile lines from different sources,and found 5 specific primers and 7 specific primers respectively to identify V and T type sterile lines.(2)There is no difference in cpDNA between the male sterile lines and their cytoplasm donors,except K‘ji5418',and because of its nature of maternal inheritance also showed no polymorphism in cpSSR amplified fragment between the male sterile lines and its fertility restoration in the F_1 generation.(3)RuBP carboxylase activity of two sets of male sterile lines was significantly higher than that of maintainer lines.In the F_1 generation,with the recovery of fertility restorer,its RuBP carboxylase activity is higher than that of maintainer lines,but lower than the respective male sterile lines in standing and flowering period.However,there is no difference between them in jointing stage.
Cytoplasmic male-sterile(CMS)lines,especially K(Aegilops kotchyi),V(Aegilops ventricosa),and T(Triticum timopheevii)types of cytoplasm,are of high value in heterosis utilization in wheat(Triticum aestivum L.).The aim of this study was to discuss the relationship between CMS and chloroplast and to reveal the mechanism of CMS by investigating chloroplast DNA and RuBP carboxylase(rubi-sco)activity in CMS male-sterile lines.The experiment was conducted with cultivars of the K,V,T-type CMS lines,the common maintainer‘Tai 911289'and‘Ji 5418',the F_1 hybrids and the cytoplasmic donors under field conditions.The major results are as follows:(1)the polymorphism between male sterile lines and maintainer lines in cpDNA,and specific fragment in the different cytoplasm type male sterile lines provides evidence of three types of cytoplasm male sterile lines from different sources,and found 5 specific primers and 7 specific primers respectively to identify V and T type sterile lines.(2)There is no difference in cpDNA between the male sterile lines and their cytoplasm donors,except K‘ji5418',and because of its nature of maternal inheritance also showed no polymorphism in cpSSR amplified fragment between the male sterile lines and its fertility restoration in the F_1 generation.(3)RuBP carboxylase activity of two sets of male sterile lines was significantly higher than that of maintainer lines.In the F_1 generation,with the recovery of fertility restorer,its RuBP carboxylase activity is higher than that of maintainer lines,but lower than the respective male sterile lines in standing and flowering period.However,there is no difference between them in jointing stage.
引文
[1]KIHARA H.Substitution of nucleus and its effects on genome manifestations[J].Cytologia,1951,16(2):177-193.
[2]高庆荣,刘保申,孙兰珍,等.K、V、A型杂种小麦细胞质效应的比较研究[J].麦类作物,1998,18(4):1-3,9.GAO Q R,LIU B S,SUN L Z,et al.Comparative study on cytoplasmic effects of K,V and A type hybrid wheat[J].Tritical Crops,1998,(4):1-3,9.
[3]张改生,杨天章.偏型、粘型和易型小麦雄性不育系的初步研究[J].作物学报,1989,15(1):1-10.ZHANG G S,YANG T Z.A preliminary on the male sterile lines of wheat with Ae.ventricosa,Ae.kotschyi and Ae.variabilis cytoplasms[J].Acta Agronomica Sinica,1989,15(1):1-10.
[4]郭瑞星,邵仁学,湖北省种子总公司.小麦杂种优势利用研究进展[J].湖北农业科学,1996,35(6):25-28.GUO R X,SHAO R X,HUBEI P S C.Advances in research on utilization of wheat heterosis[J].Hubei Agricultural Science,1996,35(6):25-28.
[5]李传友,孙兰珍.普通小麦T型,V型和K型细胞质雄性不育系花粉败育机理的细胞学研究[J].华北农学报.1996,11(2):1-8.LI C Y,SUN L Z.Cytological studies on pollen abortion mechanisms of cytoplasmic male sterile line of T,V and Ktypes in wheat[J].Acta Agireultuare Boreali-Sinica,1996,11(2):1-8.
[6]张晓科,张改生,王军卫.小麦细胞质雄性不育机理的研究进展[J].麦类作物,1997,17(2):4-7.ZHANG X K,ZHANG G S,WANG J W.Advances in research on mechanism of cytoplasmic male sterility in wheat[J].Tritical Crops,1997,17(2):4-7.
[7]乔晓琳,高庆荣,赵桂清,等.K、V、T型小麦细胞质雄性不育系籽粒的醇溶蛋白分析[J].生物技术,2006,16(1):20-22.QIAO X L,GAO Q R,ZHAO G Q,et al.Analyses of gliadins in seeds of K,V,T type cytoplasmic male sterile wheat[J].Biotechnology,2006,16(1):20-22.
[8]乔晓琳,高庆荣,张爱民,等.小麦K、V、T、CHA细胞质雄性不育类型的光合特性分析[J].作物学报,2006,32(9):1 323-1 328.QIAO X L,GAO Q R,ZHANG A M,et al.Analysis of photosynthetic characteristics of K,V,T and CHA-cytoplasmic male sterile lines of wheat[J].Acta Agronomica Sinica,2006,32(9):1 323-1 328.
[9]王永飞,马三梅,张鲁刚,等.植物细胞质雄性不育的分子机理研究进展[J].自然科学进展.2002,12(10):1 009-1 014.WANG Y F,MA S M,ZHANG L G,et al.Advances in molecular mechanism of cytoplasmic male sterility[J].Progress in Natural Science,2002,12(10):1 009-1 014.
[10]胡适宜,王模善,徐丽云.小麦雄性不育系和保持系小孢子发育的电子显微镜观察[J].北京大学学报:自然科学版,1977,(2):63-75.HU S Y,WANG M S,XU L Y.Electron-microscope observations on the microsporogenesis in male-sterile and its maintrainer-lines of wheat[J].Acta Scientiarum Naturalium Universitatis Pekinensis,1977,(2):63-75.
[11]LEVINGS C S 3RD,PRING,D R.Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasmic male-sterile maize[J].Science(New York,n.y),1976,193(4 248):158-160.
[12]HERNOULD M,SUHARSONO S,LITVAK S,et al.Male-Sterility induction in transgenic tobacco plants with an unedited apt9mitochondrial gene from wheat[J].Proceedings of the National Academy of Sciences,1993,90(6):2 370-2 374.
[13]汪月.小麦K型胞质雄性不育花药差异蛋白质组学的研究[D].山东泰安:山东农业大学,2018.
[14]李骞骞.小麦T型不育系及其保持系花药电镜观察和全蛋白的iTRAQ分析[D].山东农业大学,2015.
[15]BRYAN G J,MCNICOLL J,RAMSAY G,et al.Polymorphic simple sequence repeat markers in chloroplast genomes of Solanceous plants[J].Theoretical and Applied Genetics,1999,99(5):859-867.
[16]李继耕.叶绿体遗传与细胞质雄性不育性[J].中国农业科学,1983,16(1):49-53,97-98.LI J G.Chloroplast inheritance and cytoplasmic male-sterility[J].Scientia Agricultura Sinica,1983,16(1):49-53,97-98.
[17]侯典云,马占强,徐虹,等.小麦叶绿体DNA提取方法研究[J].河南农业科学,2011,40(10):38-40.HOU D Y,MA Z Q,XU H,et al.Study on extraction method of wheat chloroplast DNA[J].Journal of Henan Agricultural Sciences,2011,40(10):38-40.
[18]侯泽豪,张迎新,王欢,等.高温胁迫对小麦花药叶绿素质量分数及RuBP羧化酶基因表达的影响[J].西北农业学报,2018,27(9):1 280-1 286.HOU Z H,ZHANG Y X,WANG H,et al.Effects of hightemperature stress on chlorophyll mass fraction and RuBPcarboxylase gene expression in wheat(Triticum aestivum L.)[J].Acta Agriculturae Boreali-Occidentalis Sinica,2018,27(9):1 280-1 286.
[19]魏爱丽,张英华,黄琴,等.小麦不同绿色器官光合速率与碳同化酶活性及其基因型差异研究[J].作物学报,2007,33(9):1 426-1 431.WEI A L,ZHANG Y H,HUANG Q,et al.Dynamic characteristics of photosynthetic rate and carbon assimilation enzyme activities of different green organs in different genotypes of wheat[J].Acta Agronomica Sinica,2007,33(9):1 426-1 431.
[20]ISHII T,MORI N,OGIHARA Y.Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species[J].Theoretical and Applied Genetics,2001,103(6-7):896-904.
[21]李传友,谢纬武,孙兰珍,等.普通小麦三种细胞质雄性不育系线粒体DNA的比较研究[J].遗传学报,1995,(4):272-279.LI C Y,XIE W W,SUN L Z,et al.The mtDNAs’RFLP analysis of three types of cytoplasmic male sterility in wheat[J].Acta Genetica Sinica,1995,(4):272-279.
[22]李继耕.三个遗传系统及其相互渗透[M].北京:中国农业大学出版社,2000.
[23]袁健.基因组重配与重组的分子进化研究[D].安徽芜湖:安徽师范大学,2014.
[24]LI J G,LIU Y N.Chloroplast DNA and cytoplasmic malesterility[J].Theoretical and Applied Genetics,1983,64(3):231-238.
[25]刘一农,李继耕.叶绿体DNA(ctDNA)与细胞质雄性不育性[J].遗传学报,1983,10(2):114-122.LIU Y N,LI J G.Chloroplast DNA and cytoplasmic male sterility[J].Acta Genetica Sinica,1983,10(2):114-122.
[26]HE S,YU Z H,VALLEJOS C E,et al.Pollen fertility restoration by nuclear geneFrin CMS common bean:anFrlinkage map and the mode of Fraction[J].Theoretical and Applied Genetics,1995,90(7-8):1 056-1 062.
[27]孙兰珍,姚方印,李传友,等.小麦T、K、V型胞质不育系和杂种mtDNA的RAPD分析及育性相关片段的克隆[J].作物学报,2001,27(2):144-148.SUN L Z,YAO F Y,LI C Y,et al.RAPD analysing of CMS-T,K,V mtDNAs and cloning of mtDNA fragments associated with CMS-K in wheat[J].Acta Agronomica Sinica,2001,27(2):144-148.
[28]焦健,高庆荣,张爱民,等.不同小麦雄性不育类型光合、生理参数日变化的研究[J].作物学报,2007,33(8):1 267-1 271.JIAO J,GAO Q R,ZHANG A M,et al.Diurnal changes of photosynthetic and physiological parameters in different male sterile lines of wheat[J].Acta Agronomica Sinica,2007,33(8):1 267-1 271.
[29]焦健,高庆荣,王大伟,等.不同小麦雄性不育类型光合速率的影响因子分析[J].中国农业科学,2008,41(6):1 622-1 629.JIAO J,GAO Q R,WANG D W,et al.Analysis on factors affecting photosynthetic rate in different male sterility lines of wheat[J].Scientia Agricultura Sinica,2008,41(6):1 622-1 629.
[30]吴世文,张淑英,高庆荣,等.不同细胞质小麦雄性不育系杂种F1光合生理参数的分析[J].麦类作物学报,2009,29(2):217-221.WU S W,ZHANG S Y,GAO Q R,et al.Study on photosynthesis and physiological parameters in hybrids with different cytoplasmic male-sterile wheat lines[J].Journal of Triticeae Crops,2009,29(2):217-221.
[31]袁凯,高庆荣,张保雷,等.不同细胞质小麦雄性不育系及杂种F1叶绿体的观察与比较[J].中国农业科学,2012,45(10):1 887-1 894.YUAN K,GAO Q R,ZHANG B L,et al.Observation and comparison of chloroplast structure in hybrid and different cytoplasmic male-sterile wheat lines[J].Scientia Agricultura Sinica,2012,45(10):1 887-1 894.
[2]高庆荣,刘保申,孙兰珍,等.K、V、A型杂种小麦细胞质效应的比较研究[J].麦类作物,1998,18(4):1-3,9.GAO Q R,LIU B S,SUN L Z,et al.Comparative study on cytoplasmic effects of K,V and A type hybrid wheat[J].Tritical Crops,1998,(4):1-3,9.
[3]张改生,杨天章.偏型、粘型和易型小麦雄性不育系的初步研究[J].作物学报,1989,15(1):1-10.ZHANG G S,YANG T Z.A preliminary on the male sterile lines of wheat with Ae.ventricosa,Ae.kotschyi and Ae.variabilis cytoplasms[J].Acta Agronomica Sinica,1989,15(1):1-10.
[4]郭瑞星,邵仁学,湖北省种子总公司.小麦杂种优势利用研究进展[J].湖北农业科学,1996,35(6):25-28.GUO R X,SHAO R X,HUBEI P S C.Advances in research on utilization of wheat heterosis[J].Hubei Agricultural Science,1996,35(6):25-28.
[5]李传友,孙兰珍.普通小麦T型,V型和K型细胞质雄性不育系花粉败育机理的细胞学研究[J].华北农学报.1996,11(2):1-8.LI C Y,SUN L Z.Cytological studies on pollen abortion mechanisms of cytoplasmic male sterile line of T,V and Ktypes in wheat[J].Acta Agireultuare Boreali-Sinica,1996,11(2):1-8.
[6]张晓科,张改生,王军卫.小麦细胞质雄性不育机理的研究进展[J].麦类作物,1997,17(2):4-7.ZHANG X K,ZHANG G S,WANG J W.Advances in research on mechanism of cytoplasmic male sterility in wheat[J].Tritical Crops,1997,17(2):4-7.
[7]乔晓琳,高庆荣,赵桂清,等.K、V、T型小麦细胞质雄性不育系籽粒的醇溶蛋白分析[J].生物技术,2006,16(1):20-22.QIAO X L,GAO Q R,ZHAO G Q,et al.Analyses of gliadins in seeds of K,V,T type cytoplasmic male sterile wheat[J].Biotechnology,2006,16(1):20-22.
[8]乔晓琳,高庆荣,张爱民,等.小麦K、V、T、CHA细胞质雄性不育类型的光合特性分析[J].作物学报,2006,32(9):1 323-1 328.QIAO X L,GAO Q R,ZHANG A M,et al.Analysis of photosynthetic characteristics of K,V,T and CHA-cytoplasmic male sterile lines of wheat[J].Acta Agronomica Sinica,2006,32(9):1 323-1 328.
[9]王永飞,马三梅,张鲁刚,等.植物细胞质雄性不育的分子机理研究进展[J].自然科学进展.2002,12(10):1 009-1 014.WANG Y F,MA S M,ZHANG L G,et al.Advances in molecular mechanism of cytoplasmic male sterility[J].Progress in Natural Science,2002,12(10):1 009-1 014.
[10]胡适宜,王模善,徐丽云.小麦雄性不育系和保持系小孢子发育的电子显微镜观察[J].北京大学学报:自然科学版,1977,(2):63-75.HU S Y,WANG M S,XU L Y.Electron-microscope observations on the microsporogenesis in male-sterile and its maintrainer-lines of wheat[J].Acta Scientiarum Naturalium Universitatis Pekinensis,1977,(2):63-75.
[11]LEVINGS C S 3RD,PRING,D R.Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasmic male-sterile maize[J].Science(New York,n.y),1976,193(4 248):158-160.
[12]HERNOULD M,SUHARSONO S,LITVAK S,et al.Male-Sterility induction in transgenic tobacco plants with an unedited apt9mitochondrial gene from wheat[J].Proceedings of the National Academy of Sciences,1993,90(6):2 370-2 374.
[13]汪月.小麦K型胞质雄性不育花药差异蛋白质组学的研究[D].山东泰安:山东农业大学,2018.
[14]李骞骞.小麦T型不育系及其保持系花药电镜观察和全蛋白的iTRAQ分析[D].山东农业大学,2015.
[15]BRYAN G J,MCNICOLL J,RAMSAY G,et al.Polymorphic simple sequence repeat markers in chloroplast genomes of Solanceous plants[J].Theoretical and Applied Genetics,1999,99(5):859-867.
[16]李继耕.叶绿体遗传与细胞质雄性不育性[J].中国农业科学,1983,16(1):49-53,97-98.LI J G.Chloroplast inheritance and cytoplasmic male-sterility[J].Scientia Agricultura Sinica,1983,16(1):49-53,97-98.
[17]侯典云,马占强,徐虹,等.小麦叶绿体DNA提取方法研究[J].河南农业科学,2011,40(10):38-40.HOU D Y,MA Z Q,XU H,et al.Study on extraction method of wheat chloroplast DNA[J].Journal of Henan Agricultural Sciences,2011,40(10):38-40.
[18]侯泽豪,张迎新,王欢,等.高温胁迫对小麦花药叶绿素质量分数及RuBP羧化酶基因表达的影响[J].西北农业学报,2018,27(9):1 280-1 286.HOU Z H,ZHANG Y X,WANG H,et al.Effects of hightemperature stress on chlorophyll mass fraction and RuBPcarboxylase gene expression in wheat(Triticum aestivum L.)[J].Acta Agriculturae Boreali-Occidentalis Sinica,2018,27(9):1 280-1 286.
[19]魏爱丽,张英华,黄琴,等.小麦不同绿色器官光合速率与碳同化酶活性及其基因型差异研究[J].作物学报,2007,33(9):1 426-1 431.WEI A L,ZHANG Y H,HUANG Q,et al.Dynamic characteristics of photosynthetic rate and carbon assimilation enzyme activities of different green organs in different genotypes of wheat[J].Acta Agronomica Sinica,2007,33(9):1 426-1 431.
[20]ISHII T,MORI N,OGIHARA Y.Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species[J].Theoretical and Applied Genetics,2001,103(6-7):896-904.
[21]李传友,谢纬武,孙兰珍,等.普通小麦三种细胞质雄性不育系线粒体DNA的比较研究[J].遗传学报,1995,(4):272-279.LI C Y,XIE W W,SUN L Z,et al.The mtDNAs’RFLP analysis of three types of cytoplasmic male sterility in wheat[J].Acta Genetica Sinica,1995,(4):272-279.
[22]李继耕.三个遗传系统及其相互渗透[M].北京:中国农业大学出版社,2000.
[23]袁健.基因组重配与重组的分子进化研究[D].安徽芜湖:安徽师范大学,2014.
[24]LI J G,LIU Y N.Chloroplast DNA and cytoplasmic malesterility[J].Theoretical and Applied Genetics,1983,64(3):231-238.
[25]刘一农,李继耕.叶绿体DNA(ctDNA)与细胞质雄性不育性[J].遗传学报,1983,10(2):114-122.LIU Y N,LI J G.Chloroplast DNA and cytoplasmic male sterility[J].Acta Genetica Sinica,1983,10(2):114-122.
[26]HE S,YU Z H,VALLEJOS C E,et al.Pollen fertility restoration by nuclear geneFrin CMS common bean:anFrlinkage map and the mode of Fraction[J].Theoretical and Applied Genetics,1995,90(7-8):1 056-1 062.
[27]孙兰珍,姚方印,李传友,等.小麦T、K、V型胞质不育系和杂种mtDNA的RAPD分析及育性相关片段的克隆[J].作物学报,2001,27(2):144-148.SUN L Z,YAO F Y,LI C Y,et al.RAPD analysing of CMS-T,K,V mtDNAs and cloning of mtDNA fragments associated with CMS-K in wheat[J].Acta Agronomica Sinica,2001,27(2):144-148.
[28]焦健,高庆荣,张爱民,等.不同小麦雄性不育类型光合、生理参数日变化的研究[J].作物学报,2007,33(8):1 267-1 271.JIAO J,GAO Q R,ZHANG A M,et al.Diurnal changes of photosynthetic and physiological parameters in different male sterile lines of wheat[J].Acta Agronomica Sinica,2007,33(8):1 267-1 271.
[29]焦健,高庆荣,王大伟,等.不同小麦雄性不育类型光合速率的影响因子分析[J].中国农业科学,2008,41(6):1 622-1 629.JIAO J,GAO Q R,WANG D W,et al.Analysis on factors affecting photosynthetic rate in different male sterility lines of wheat[J].Scientia Agricultura Sinica,2008,41(6):1 622-1 629.
[30]吴世文,张淑英,高庆荣,等.不同细胞质小麦雄性不育系杂种F1光合生理参数的分析[J].麦类作物学报,2009,29(2):217-221.WU S W,ZHANG S Y,GAO Q R,et al.Study on photosynthesis and physiological parameters in hybrids with different cytoplasmic male-sterile wheat lines[J].Journal of Triticeae Crops,2009,29(2):217-221.
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