植物细胞质雄性不育系育种的反向核置换技术分析

通过不育细胞质为选择背景,在田间事先鉴定出杂种后代的优异完全不育株,用花药培养或诱导孤雌生殖使其纯合,测定其配合力,可以筛选到优良的目标不育系.以下3种方法则可能通过目标不育系而获得其同型保持系:一是通过体细胞变异(花药培养)产生;二是在不育系孕穗期高温或低温处理使其转换成可育,选择仍具有不育保持能力的作为保持系,或作为轮回亲本,将其细胞核换到可育细胞质中;三是用原生质体融合的方式向不育系导入已杀死细胞核的可育细胞质而获得配套保持系.它可以使杂种优势利用变得有预见性,可能提高现有杂种优势水平.在创造雄性不育新质源,排除微效可育基因,进行不育系的定向改造,选育高配合力不育系,以及加速育种进程等方面具有重要价值.     

大白菜细胞质雄性不育的分子生物学研究

细胞质雄性不育(cytoplasmic male sterility,CMS)是作物杂种优势利用的一 条重要途径。对其机制的研究不仅具有重要的理论意义,而且具有重要的应用价值。对大白菜而言,利用CMS最关键的环节是要育成不育系和其相应的保持系。利用不育系和其它材料杂交产生F1代种子;用不育系和保持系杂交来繁殖不育系;保持系自交繁殖种子。但采用常规的方法来选 育不育系和其相应的保持系周期长、见效慢,远不能满足生产发展的需要。现代生物技术为创造新的不育系和保持系提供了便利。本论文对大白菜细胞质雄性不育的分子机理和 RAPD分析的有关问题进行了研究。     

叶用芥菜细胞质雄性不育系0912A的胞质效应和杂种优势分析

通过异地多代的育性鉴定及花器形态观察来调查叶用芥菜细胞质雄性不育系0912A的不育性及结籽能力;同时分别以不育系0912A和其保持系0912B为母本,与5个典型叶用芥菜自交系配成10个同核异质的杂交种,以研究hau胞质的胞质效应并测定杂种优势.结果显示:该不育系败育彻底稳定,不育株率和不育度均为100％,有正常的结籽能力;不育系杂种一代与产最相关性状的杂种优势明显,但品质性状无优势;hau胞质在单株重上的负效应较明显,在其他性状上的负效应相对较弱,但hau胞质组合F1的单株重杂种优势依然显著,且胞质效应受杂交父本核影响,通过选择合适的杂交父本配组,细胞质的负效应可以减轻或消除.     

青花菜两类雄性不育系花器官形态结构的比较

青花菜是一种重要的十字花科蔬菜作物,具有良好的抗癌功效。随着青花菜栽培面积的不断扩大和抗癌药物的开发,对青花菜种子的需求量也不断增加。利用雄性不育系生产青花菜F1杂交种子是其杂种优势利用的重要途径,但常用的细胞质雄性不育系在生产中存在前期死蕾较重、花蜜量少和种子产量不高等问题。本课题组用优良的青花菜自交系为父本,以甘蓝显性细胞核雄性不育材料79-399-3和细胞质不育材料OguraCMSR3629为不育源,通过回交转育的方法获得了多个青花菜显性细胞核雄性不育系和细胞质不育系,为了进一步有效利用其青花菜雄性不育系,寻求更好的不育源。本研究以青花菜高代自交系(保持系)8554、8590、93219及由其经多代回交转育而成的两类雄性不育系为试材,对其死蕾数、花器官形态和蜜蜂访花情况进行了比较研究。结果表明,在花蕾长、花蕾直径、单枝死蕾数、花冠直径、雄蕊长、花瓣长、花药长、蜜蜂访花次数、来访蜜蜂数及访花时间等方面,经相同自交系转育而成的显性细胞核雄性不育系(DGMS)与细胞质雄性不育系(CMS)间的差异有统计学意义,总体表现为DGMS优于CMS。     

在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100％不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。     

玉米新选细胞质雄性不育系小孢子发育的细胞学观察及DNA甲基化分析

细胞质雄性不育在高等植物中普遍存在,是杂种优势利用的重要工具,为推动植物杂种优势的利用发挥了重要作用。文章以本课题组前期新选的玉米细胞质雄性不育系A1、A2及保持系18(红)为材料,利用石蜡切片技术对不育材料小孢子发育过程进行细胞学观察,采用高效液相色谱法(HPLC)对不同发育时期的叶片及不同发育时期的雄穗DNA进行甲基化分析,从细胞学和表观遗传学角度了解不育系A1、A2的败育机制。结果表明：不育材料A1、A2小孢子发生败育的主要时期为四分体时期至单核小孢子中期。在不育系A2中还存在另一种败育方式,即在花粉母细胞时期表现出败育特征。甲基化分析结果表明,保持系18(红)的叶片DNA甲基化水平从苗期到拔节期迅速上升,而不育系A1、A2叶片DNA甲基化水平基本保持不变;保持系雄穗DNA甲基化水平表现为从花粉母细胞时期到双核期逐渐升高,而不育材料A1、A2从花粉母细胞时期到双核期的雄穗DNA甲基化水平表现为先上升后下降的趋势,达到最高峰的时期均出现在小孢子发育的四分体时期。从小孢子发育的细胞学观察结果可以发现,小孢子败育的主要时期往往具有较高的甲基化水平,推测DNA甲基化水平变化可能与不育材料A1、A2的花粉败育有关。     

在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100%不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。     

小麦不同细胞质雄性不育系及其保持系萌动胚及幼芽可溶性蛋白等电…

用等电聚焦电泳分析的方法,测定了小麦３种细胞质雄性不育类型（Ａ型、Ｅ型、Ｔ型）及其相应同核保持系萌动胚及动幼芽可溶性蛋白。发现雄性可育系等电点（ｐＩ）为４．９０的蛋白质成数量高于相应的不育系;ｐＩ为６．８５的蛋白质可能是Ｔ型细胞质基因表达的结果;ｐＩ为７．６的蛋白质可能为津丰Ａ不育系特有的区带。表明细胞质来源不同的不育类型,其萌动胚及动芽可溶性蛋白等电聚焦电泳图谱差异明显,有可能作为鉴别它们的依据     

小麦不同细胞质雄性不育系及其保持系萌动胚及幼芽可溶性蛋白等电聚焦电泳分析

用等电聚焦电泳分析的方法,测定了小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）３ 种细胞质雄性不育类型（Ａ 型、Ｅ型、Ｔ型）及其相应同核保持系萌动胚及幼芽可溶性蛋白． 发现雄性可育系等电点（ｐＩ）为４．９０ 的蛋白质合成数量高于相应的不育系;ｐＩ为６．８５ 的蛋白质可能是Ｔ 型细胞质基因表达的结果;ｐＩ为７．６ 的蛋白质可能为津丰Ａ 不育系特有的区带．表明细胞质来源不同的不育类型,其萌动胚及幼芽可溶性蛋白等电聚焦电泳图谱差异明显,有可能作为鉴别它们的依据     

玉米三系及其杂种根尖染色体C-带带型的遗传分析

用玉米的两个雄性不育系,和它们共同的保持系、恢复系以及两个不育系和恢复系杂交的F_1进行了根尖染色体C-带带型的遗传分析。从明显的C-带带型看,不育系与保持系相同,恢复系与不育系和保持系显著不同。在F_1中,从父母本之间带型有明显差异的4对染色体看,每对同源染色体的一条,其带型和一个亲本相似,另一条和另一个亲本相似。以明显的C-带带型作为标记,在杂交后代中可以直接对不同亲本遗传下来的染色体进行追踪。 文中讨论了在明显C-带带型方面细胞质对细胞核有无影响,用品种间明显C-带带型的差异大小作为选配强杂种优势组合的依据,以及从细胞学上鉴定核代换程度的标准的可能性。     

两份太空诱变玉米雄性不育突变体的遗传研究

从搭载神舟4号飞船的4份玉米自交系后代中选育出两份雄性不育突变体, 对其进行育性鉴定, 并分析不育性状的稳定性及遗传特点。以不育材料的不育株为母本, 同群体的可育株和其他自交系为父本进行杂交, 结合自交、回交分析其后代的育性表现; 同时, 以具有正常细胞质的自交系为母本, 育性完全恢复的测交F1植株为父本进行反交, 对其反交的F1及F2进行育性观察分析。结果表明：这两份不育材料不育株的花药内无花粉或含少量畸形花粉, 败育彻底, 花粉败育表现为典败型。不育性状在不同年份、不同季节、不同地点下稳定遗传, 属可遗传的单基因控制的隐性核不育类型。     

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.     

Differential Mitochondrial Electron Transport through the Cyanide-Sensitive and Cyanide-Insensitive Pathways in Isonuclear Lines of Cytoplasmic Male Sterile, Male Fertile, and Restored Petunia

Three pairs of isonuclear lines of cytoplasmic male sterile (CMS) and fertile Petunia cells (Petunia hybrida [Hook] Vilm. and Petunia parodii L.S.M.) grown in suspension culture were examined for sensitivity to inhibitors of respiratory electron transport at time-points after transfer into fresh media. Cells from CMS lines differed from cells of fertile lines in their utilization of the cyanide-insensitive oxidase pathway. Under our culture regime, after approximately 3 days of culture cells from the CMS lines exhibited much lower cyanide-insensitive, salicylhydroxamic acid-sensitive respiration than cells from the fertile lines. This respiratory difference was shown to be specific to the mitochondrial alternative oxidase pathway by using other characteristic inhibitors of mitochondrial electron transport in experiments with isolated mitochondria. Immature anthers from CMS plants also showed lower alternative oxidase activity relative to anthers from male fertile plants, but no such difference was detected in leaf tissue, ovary or perianth tissue, or anthers collected just prior to anthesis. A cell line from a fertile plant carrying a nuclear fertility restorer gene and the CMS cytoplasm exhibited increased activity of the alternative pathway compared with the CMS lines.     

提高光敏感雄性不育水稻花粉单倍体育种效率及不育花粉植株育性转换特点的研究

比较了(光敏s/正常品种)F_1及F_2为供体亲本,对在花药培养时所获得的花粉植株中不育个体/全部花粉植株之比例的影响。结果表明,以F_1为供体亲本,在所获得的二倍体花粉植株(A_1)中,不育株(长日下)约占20%左右;而从F_2分离的不育株为供体亲本,相应的比例为90%左右。对获得不育的花粉植株而言,供体亲本经过F_2的选择,在花粉一代中可以提高育种效率3—4倍。指出,以培育光敏感雄性不育系为目的的花药培养,与一般育种之花药培养采用杂种F_1为供体亲本不同,不仅应对杂种F_2代在长日照条件下进行不育株的选择,而且应在短日照下对这种不育株作育性转换的双重选择。以这种个体作为花药培养的供体亲本,可以大大提高育种效率。 在长日照下表现不育的花粉植株的育性转换具多样性。来自同一组合的不育花粉植株在晚造(短日照)条件下,其花粉有的染色,频率高且稳定;有的虽然可变为染色,但频率不高或不稳定或二者兼有;有些却一直不为Ⅰ-KⅠ染色,或即使染色频率也在10%以下。这一结果与收集全国各地15个光敏核不育系在本昕同期种值条件下的反应十分吻合。这说明通过花药培养,从特定的组合培育出所需要的光敏/光温互作或温敏型的核不育系的可能性是存在的。     

A ras GTPase-activating protein-binding protein, TaG3BP, associated with the modulation of male fertility in a thermo-sensitive cytoplasmic male sterile wheat line

Hybrid wheat development may contribute to higher, more stable yield and could result in greater food security for much of the world's growing population. YS type thermo-sensitive cytoplasmic male sterile (TCMS) wheat lines were developed for use in hybrid wheat breeding in China. To investigate the molecular mechanism of modulation of male fertility in the YS-type TCMS wheat lines, a ras GTPase-activating protein-binding protein (TaG3BP) was examined. The deduced amino acid sequence encoded by TaG3BP was conserved in the sequenced genomes of Embryophyte. TaG3BP expression in the anthers of YS-type TCMS lines taken at the critical fertility reversion stage of pollen development from male fertile anthers was higher than that from male-sterile anthers, either by quantitative real-time PCR or by western blot analysis. Sequence analysis on the cDNA and genomic DNA of TaG3BP in three kinds of K-type CMS wheat lines and their maintainers indicated that there were no significant difference between the genes or in their 5' flanking sequences. The TaG3BP expression revealed by quantitative real-time RT-PCR was lower in the young spikes of these CMS lines than that of their maintainers. This indicates that TaG3BP expression is associated with the modulation, from male-sterile to fertile, of the TCMS wheat line, and TaG3BP might be a key factor in the pathway responsible for the fertility reversion.     

萝卜细胞质雄性不育恢复基因的RAPD标记

以萝卜恢复系9802和不育系9802A配制杂交组合,并以174株个体组成的F2分离群体作为恢复基因的标记群体.以分离群体的不育株和可育株分别建立不育池和恢复池,利用100个RAPD引物对两池间的多态性进行研究.分析表明引物OPC6在两池间扩增出稳定的多态性差异.经连锁分析,证明标记OPC61900与萝卜细胞质雄性不育恢复基因连锁,遗传距离为11.6cM(Centimorgan).这个标记可应用于对育性恢复基因的标记辅助选择.     

水稻雄性不育系及雄性不育突变体筛选研究进展

 为了获得新细胞质源的水稻雄性不育系及雄性不育突变体,以解决目前杂交水稻生产上所面临的不育系来源单一的问题,人们采用了常规育种、诱变育种、组织培养和离体诱变等方法,其中离体筛选尤显其优势性,但也有不少问题有待解决,如能通过离体培养定向地筛选出雄性不育系及雄性不育突变体,无疑将大大提高工作效率。此外,对影响离体筛选雄性不育突变体的因素进行了探讨。     

DNA synthesis and cytoplasmic differentiation in tapetal cells of normal and cytoplasmically male sterile lines of Petunia hybrida

Summary A new method is described by which tapetal cells may be isolated from anthers of cytoplasmic male sterile (CMS) and fertile lines of Petunia hybrida. Using a combination of stereometry and Feulgen densitometry it has been possible to chart DNA synthesis and nuclear division with great precision within these cells. Results are presented which show CMS lines both to synthesize far less DNA than the fertiles and also to undergo less cell division. These differences in DNA kinetics and cytokinesis are obvious long before any differences between sterile and fertile lines may be detected in the meiocyte mass. In addition to these difference in nuclear behaviour, the tapetal cells of CMS lines also synthesize far lower levels of rough endoplasmic reticulum than do their fertile counterparts. Again, this difference is evident at a very early stage of anther development when all other cell components, including mitochondria and plastids, appear normal. These data are discussed in terms of the very special differentiation undergone by tapetal cells in angiosperms, and the conclusions drawn are considered in the perspective of current hypotheses proposed to explain the operation of CMS.