玉米CMS-S小孢子败育过程中的细胞程序性死亡

以玉米（Zea mays L.）S型细胞质雄性不育系(CMS-S)的一对近等基因系S-Mo17^Rf3Rf3 和S-Mo17^rf3rf3为材料，采用TdT介导的dUTP DNA末端标记（TUNEL）、细胞色素C免疫原位杂交和DNA寡聚核小体片段电泳等方法，分别在细胞学水平和DNA水平上研究了玉米CMS-S小孢子败育的细胞程序性死亡（PCD）过程。结果表明，在花粉母细胞减数分裂后的四分体解离时期，不育花药的绒粘层细胞较可育花药提前裂解；在不育系S-Mo17^rf3rf3花药和花粉S-rf3中均明显出现PCD过程的DNA片段化以及线粒体细胞色素C外渗的现象，证明了玉米CMS-S的花粉败育与花药绒粘层细胞的提前凋亡和小孢子细胞的程序性死亡有关。     

Cytological screening of rice TGMS lines

An investigation was undertaken to develop new stable thermo‐sensitive genic male sterility (TGMS) rice lines in intermated progenies (IMPs) of TGMS lines by using an anther clearing technique. The results indicate that both pre‐ and post‐meiotic genetic systems operate during anther development for the expression of sterility in TGMS lines. In all the TGMS lines, sterile anthers were small with empty pollen grains of irregular shape, except for TS 16, which showed pollen‐free anthers. This indicates that the sensitive stage of TS 16 is around stage IV (stamen and pistil primordia) of panicle development. Distinct differences were observed between sterile and fertile phases with respect to anther size, shape and colour of the pollen grains in TS 18 and TS 29. The pollen grains at the sterile phase were small and irregular in shape while in the fertile phase they were plump and larger with a yellow colour, establishing that the occurrence of sterility in TS 18 and TS 29 is post‐meiotic. Three distinct classes of pollen fertility percentage viz. <20%, 50‐70% and >90% were observed in IMPs. Anther clearing in IMPs showed distinct developmental patterns of pollen production with respect to distinct classes of pollen fertility. Less than 20% pollen fertility was observed in hybrids such as TS 15 × TS 16, TS 15 × Co 47, TS 18 × TS 16 and TS 18 × Co 47 which hold promise for developing new TGMS lines with a good plant type and acceptable quality.     

Water Deficit Reduced Fertility of Young Microspores Resulting in a Decline of Viable Mature Pollen and Grain Set in Rice

Pollen formation in rice ( Oryza sativa L.) is highly vulnerable to environmental stresses such as heat, chilling and drought. In rice plants exposed to drought during male reproductive development, the most obvious damage often observed is a decline in the number of engorged pollen and grain set. This has been well characterized in rice under chilling and to a lesser extent under drought stress. Moreover, detailed literature on the immediate effects of drought on developing young microspores in rice is still limited. Here, we report findings from experiments on rice plants exposed to water deficit for three consecutive days during early stages of anther development. When the osmotic potential of the growing medium was equal to or less than −0.5 MPa, as induced by polyethylene glycol, the leaf water potential was significantly lowered and grain set was reduced. A strong correlation between grain set and viable young microspores (P < 0.001, r² = 0.8223) indicates that water deficit immediately reduced fertility of rice plants at the time of exposure. This result suggests a new underlying mechanism of water deficit-induced pollen abortion in rice.     

黏类小麦细胞质雄性不育相关基因cMDH的克隆与表达分析

为深入研究黏类小麦雄性不育的分子遗传机制,利用抑制差减杂交技术构建了黏类小麦育性相关基因的二核期SSH文库.经文库筛选后,得到一个在可育文库中表达的与胞质苹果酸脱氢酶基因同源的EST序列.以该EST序列为信息探针,经电子克隆、RT-PCR、PCR克隆与序列分析,获得了小麦胞质苹果酸脱氢酶(cytosolic malate dehydrogenases,cMDH)基因的cDNA与DNA序列,利用荧光定量PCR技术对该基因在不育株和可育株花药中的表达进行了分析,并比较了MDH在小麦不育株和可育株中的活性变化.结果表明,该基因的cDNA序列长1213 bp,编码333个氨基酸;DNA序列长2 908 bp,含有7个外显子和6个内含子;该基因在不育株和可育株花药发育3个时期(单核、二核和三核)的表达均表现为先升后降的模式,而且该基因在不育株花药发育的二核期和三核期的表达相对于可育株被明显抑制;MDH在小麦不育株和可育株中的活性变化趋势与定量结果一致.推测该基因在花粉发育早期表达,它的下调表达可能影响了小麦雄蕊发育过程中的能量供应而导致雄性不育.     

洋葱63A细胞质雄性不育与绒毡层的提早衰退有关

细胞质雄性不育（CMS）机理的细胞学研究在许多作物上已有报道，如拟南芥、大豆、水稻、小麦等，但在国内至今未见关于洋葱的此类报道。利用光镜和透射电镜对洋葱不育系63A和保持系63B进行花药发育过程中显微结构和超微结构观察，结果表明，不育花药花粉母细胞时期，花粉母细胞发育正常，花粉囊形状不规则，绒毡层发育迟缓。四分体时期，四分体形成正常，中层严重退化，绒毡层与药室壁完全脱离，细胞质浓缩、空泡化。利用DNA梯度技术发现不育系绒毡层细胞提早发生程序性死亡。小孢子发育时期，小孢子细胞质发生浓缩、降解，绒毡层完全解体。推测不育系小孢子败育与中层、绒毡层提前衰退有关。     

Genic analysis of hybrid sterility caused by anther indehiscence between distantly related rice varieties

Hybrid sterility of the cross between Silewah, an Indonesian native variety and Hayakogane, a Hokkaido rice variety in Japan was confirmed to be caused by anther indehiscence, based on a lot of spikelets with fewer numbers than 3 of dehiscent anthers and fewer numbers of pollens poured onto stigma per spikelet in Silewah/Hayakogane F1. In Silewah/Hayakogane//Hayakogane BC₁F₁, spikelet fertility was correlated with mean number of dehiscent anthers per spikelet. So, genic analyses for hybrid sterility by anther indehiscence were conducted by scoring spikelet sterility in F₂, BC₁F₁s and triple cross. As a result, it was concluded that the hybrid sterility was controlled by complementary action of three dominant genes. Silewah has one of the three genes and Hayakogane has the other two. It was presumed that the two genes which Hayakogane possesses were derived from those of Aikoku, an old native variety in Japan. This revised version was published online in July 2006 with corrections to the Cover Date.     

光周期敏感核不育水稻农垦58S不育花药的显微结构变化

本文观察了光敏感核不育水稻农垦58S 在小孢子发生和发育过程中不育和可育花药的显微结构差异。结果表明,不育花药在小孢子母细胞时期和减数分裂期,药隔维管束无导管和筛管,在单核晚期,有分化较差的导管和筛管,其薄壁细胞发育不良或退化,鞘细胞皱缩,由绒毡层的细胞质构成的原生质团不解体。药隔维管束发育异常和绒毡层延迟解     

DROUGHT STRESS: Role of Carbohydrate Metabolism in Drought‐Induced Male Sterility in Rice Anthers*

Rice plants exposed to three consecutive days of water stress (?0.5 MPa) show a reduction in male fertility and grain set, which is attributed to increased levels of reactive oxygen species (ROS) and activation of a programmed cell death. This current research was conducted to further investigate the association of sugar metabolism with microspore abortion in rice anthers. Biochemical assays showed that sucrose, glucose and fructose contents were found to be significantly increased in anthers from water stressed plants compared with the control. qRT‐PCR analyses and in situ hybridization of metabolic genes (sugar transporters, invertase and phosphotransferase/kinases) demonstrated that the supply of sugars for developing microspores and the initial steps of sugar utilization e.g. glycolysis, were not repressed. However, it appears that the accumulation of sugars in stressed anthers might involve a reduction of mitochondrial activity during the tricarboxylic acid cycle, which could result in excessive production of ROS and a depletion of the ATP pool. These results also suggest that higher levels of sugars at all stages of anther development seemed to be associated with some measure of protection to the anthers against oxidative stress. Induced expression of sugar transporter genes might have maintained the high levels of sugar in the tapetum and the locules, which alleviated oxidant damage caused by excessive ROS generation. Thus, the increased level of sugars might potentially be a natural response in providing protection against oxidant damage by strengthening the antioxidant system in anthers.     

白菜型冬油菜温敏不育系PK3-12S育性转换的差异蛋白质组学分析

为揭示温敏不育系PK3-12S育性转换机制,本研究以白菜型冬油菜温敏不育系PK3-12花药为材料,采用2-DE和LC-MS/MS质谱鉴定等差异蛋白组学方法,分离鉴定了PK3-12在不育/可育条件下花药差异表达蛋白质,并对差异表达蛋白进行了生物信息学分析;进而采用RT-PCR检测了PK3-12在不育/可育条件下花蕾发育进程中差异蛋白编码基因表达量变化。结果表明,高温不育条件下, PK3-12花药形态瘪小,药室有少量败育花粉,育性转换受1对隐性基因控制,表达变化量在2倍以上差异蛋白质点31个,其中增量表达蛋白质点6个,减量表达蛋白质点11个,表达完全抑制蛋白点12个,不育花药特异表达蛋白点2个。质谱鉴定出15个差异蛋白质,参与信号转导通路、二羧基乙醛酸代谢、糖酵解代谢、次生合成代谢、氨基酸生物合成、分支酸生物合成、碳代谢途径等细胞过程。Rubisco亚基连接蛋白编码基因BrrbcL开放读码框(open reading frame, ORF)长度为1095 bp,编码364个氨基酸;与可育花蕾相比,发育进程中不育花蕾BrrbcL基因、膜联蛋白基因(ANN)、BetVI过敏原家族基因(BetV...     

棉花细胞质雄性不育花药败育过程中内源激素的变化

利用酶联免疫检测（ELISA）技术,研究了棉花细胞质雄性不育系、保持系、恢复系和杂种一代花药在造孢细胞增殖期、花粉母细胞减数分裂期、四分体至小孢子释放期、花粉发育期和花粉成熟期内源吲哚乙酸（IAA）、赤霉素（GA₃）、玉米素核苷（ZR）和脱落酸（ABA）含量的动态变化。结果表明,在保持系、恢复系和杂种一代可育花药间内源激素含量差异不明显,但在可育与不育花药间差异显著。在IAA、GA₃和ZR含量上,不育花药低于可育花药,但在ABA含量上,不育花药高于可育花药,这种差异在花粉母细胞减数分裂时期达最大值。根据内源激素的功能推测,不育花药IAA含量过低使花药淀粉积累受阻,GA₃含量不足影响花粉母细胞和绒毡层细胞的膨大,过低的ZR含量使花粉母细胞不能分裂形成四分体,过高的ABA含量促进花粉母细胞退化和死亡。     

Identification of Physiological Traits Underlying Cultivar Differences in Drought Tolerance in Rice and Wheat

Rice is used as a model cereal to study drought response at the molecular level, with the goal of applying results to other cereals. To assess the relevance of results from rice to other species, the kinetics of drought development and plant response of tolerant and susceptible tropical rice ( Oryza sativa L.) and subtropical wheat ( Triticum aestivum L.) cultivars were compared under vegetative and reproductive stage drought in pot experiments. Water was withheld during reproductive stage until plant available soil moisture content was 30 % of field capacity (FC) or leaf wilting was observed, and then reapplied. Rice reached 30 % FC 9 days after withholding water and wheat after 13 days. Before rewatering, both species reached leaf water potentials of −12 bars and similarly low transpiration rates. Stress reduced leaf relative water content, leaf elongation and membrane stability. When water stress was imposed during reproductive stage, pollen fertility was most affected in wheat, while panicle exsertion and anther dehiscence were severely affected in rice. When water stress was imposed during vegetative stage, wheat was less affected to vegetative stage drought than rice. The nature of differences between tolerant and susceptible cultivars was similar for the two species. However, the differential growth habitats and growth rate of plants needs to be considered in these kinds of experiments.     

新型陆地棉细胞质雄性不育系花器形态学和细胞学观察

 采用石蜡切片法,对一种来源于陆地棉的新型细胞质雄性不育材料的3份转育材料（G22A,H109A和1793A）的花药发育过程进行了花器形态及细胞学观察。花器形态和细胞学观察结果表明,G22A,H109A和1793A三个不育系均花瓣小,花丝短,柱头明显外露,败育彻底,无花粉。败育时期主要发生在造孢细胞增殖时期至小孢子母细胞减数分裂前,表明这3份不育材料不育性状不受细胞核基因的影响。对不育材料与其保持系细胞学观察发现,不育材料与正常花药发育细胞学形态上主要差别是绒毡层发育。不育系绒毡层的着色不明显,大小与中层细胞相似且在整个花药发育过程中不降解,而保持系绒毡层的着色深,在小孢子发育过程中逐渐降解。因此,根据细胞学研究结果可以说明花药中绒毡层发育异常是该来源于陆地棉的新型细胞质雄性不育材料败育的细胞学基础。     

水分胁迫对水稻结实期一些生理性状的影响

选用抗旱性不同的3个杂交水稻品种，在干旱棚内分别在正常供水和水分胁迫条件下，研究了水稻结实期丙二醛（MDA）含量、光合速率、气孔导度、渗透调节物质及保护性酶活性等生理性状变化。结果表明，结实期随土壤水分胁迫加剧和时间延长细胞膜脂过氧化加剧，抗旱性弱的品种膜脂过氧化更为严重；剑叶光合速率、气孔导度和叶绿素含量迅速下降，抗旱性弱的品种比抗旱性强的品种降速快、幅度大。抽穗后0～14 d随胁迫时间的延长叶片内可溶性糖、氨基酸含量增加，超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）3种酶活性升高，抗旱性强的品种增加或升高的幅度大。抽穗14 d后叶片内可溶性糖、氨基酸含量下降，SOD、CAT和POD酶活性降低。试验表明，水稻叶片MDA含量、光合作用、渗透调节物质及保护性酶活性的变化幅度与品种的抗旱性密切相关。     

Cajanus platycarpus (Benth.) Maesen as the donor of new pigeonpea cytoplasmic male sterile (CMS) system

Cajanus platycarpus, a distantly related wild species, was successfully crossed with cultivated pigeonpea using embryo rescue and tissue culture techniques. Advance generation lines showed a range of desirable characters including cytoplasmic male sterility. A range of pigeonpea cultivars restored fertility and was maintained by a few lines including cultivar ICPL 85010. Clasmogamous flowers were observed in the male sterile lines. In such flowers anthers did not form di-adlephous bundle. Cytological analysis revealed that meiosis proceeded normally till the tetrad stage in those anthers with pollen grains. After which many of the pollen grains turned sterile. In the anthers with pollen grains, dehiscence was not observed, thus creating functional sterility. In many other anthers, pollen mother cells (PMCs) were not formed at all, giving rise to sepalous anthers. In conclusion two mechanisms of male sterility existed, one was premeiotic, where PMCs did not form and in the second, although PMCs gave rise to pollen grains, they were either partially or totally sterile accompanied by non-dehiscence of anther wall.     

玉米雄性不育细胞质细胞色素氧化酶活性及ATP含量的研究

研究了玉米ＭＯ１７背景三种雄性不育细胞质（Ｔ．Ｃ．Ｓ）及正常细胞质（Ｎ）系小孢子发育过程中花药组织细胞色素氧化酶活性,ＡＴＰ含量及其变化。发现各不育胞质细胞素氧化酶活性及ＡＴＰ含量的下降。讨论了玉米雄性不育细胞质的能量亏损现象。     

New insights into genotypic thermodependency of cytoplasmic male sterility for hybrid barley breeding

The cytoplasmic male sterility (CMS) system msm1 in barley is known to be thermosensitive, sometimes resulting in spontaneous fertility restoration in the absence of the corresponding restorer gene Rfm1. Here, we investigated genotypic differences concerning temperature sensitivity and the plant developmental stage at which elevated temperature induces spontaneous fertility restoration in three CMS mother lines. While one line stayed completely male sterile, a significantly higher fertility was observed in two lines after treatment from growth stage DC 41 until maturation. Microscopic analysis revealed that sterile anthers contained neither intact pollen, nor remains of aborted pollen grains, whereas pollen was visible in anthers of potentially fertile plants. We conclude that the barley CMS system affects anther and pollen development prior to meiosis. Elevated temperature during heading and flowering can lead to a spontaneous fertility restoration by reactivating pollen growth. Nevertheless, genotypic variation exists enabling the selection for stable CMS mother lines and the development of F₁ hybrids with high hybridity. As spontaneous fertility restoration due to environmental effects is difficult to phenotype, further investigations will focus on the development of molecular markers for marker‐assisted selection.     

Influence of water availability on fertility restoration of CMS lines with the 'M35', A4 and '9E' CMS-inducing cytoplasms of sorghum

Male fertility restoration in new types of sorghum cytoplasmic male sterility‐inducing cytoplasms (A4, ‘9E’, ‘M35’), characterized by the formation of non‐dehiscent anthers, is difficult. Lines with fertility‐restorer genes for these unique cytoplasms do occur, but rarely, and when found tend to be unstable in their inheritance and expression. The aim of this research was to explore reasons for this instability. Seven lines in three unique cytoplasms, ‘9E’, A4 and ‘M35’, and six lines that restore with these cytoplasms were grown at the Agricultural Research Institute for South‐East Region in Saratov, Russia from 1993 to 2004. Levels of male fertility restoration and various environmental factors were recorded. It is reported that for sorghum hybrids in the A4, ‘9E’ and ‘M35’ male‐sterile cytoplasms, the level of plant male fertility is determined by the level of water available to plants during anther and pollen formation that which ‘switches on’ the expression of fertility‐restoring genes, and is possibly involved in an unusual type of male fertility inheritance in these cytoplasms. The creation of reliable line‐fertility restorers capable of the restoration of male fertility of F₁ hybrids in ‘M35’ cytoplasm under conditions of water stress is also reported. Current research explore mechanisms involved possible in responses to water levels at various growth stages and their influence on fertility within these cytoplasms.     

湖南水稻干旱评估方法研究

为了探讨双季早稻、双季晚稻、一季稻干旱影响定量评估方法,统计各熟制水稻各生育期综合气象干旱指数、水分亏缺指数与水稻受旱率、成灾率、单位面积粮食减产量的相关,分析各熟制水稻干旱影响敏感期;基于相关系数,定量计算综合气象干旱指数、水分亏缺指数组合成综合评估指标所占的权重,组建各熟制水稻旱情综合评估指标;基于各熟制水稻旱情综合评估指标,结合抗旱能力,通过多元回归建立各熟制水稻受旱率、成灾率、单位粮食减产量评估模型。结果表明：（1）湖南季节性干旱特征明显,对于双季晚稻,各生育期干旱出现频率基本接近,干旱影响期为返青分蘖期和幼穗形成期;双季早稻、一季稻各生育期干旱出现频率差异较大,以水稻生长后期干旱出现频率高,而水稻干旱影响敏感期也在水稻生长后期。（2）双季早稻干旱灾害评估模型历史同级拟合率平均为77.6%,级差一级以下的拟合率为91.1%;双季晚稻同级拟合率为70.9%,级差一级以下的拟合率为83.0%;一季稻同级拟合率为61.8%,级差一级以下的拟合率为84.5%。基于降水、需水、可供水等因子建立水稻干旱灾害评估模型,物理意义清楚,准确率较高,可为水稻干旱影响定量评估提供技术支持。     

培养基和基因型对粳灿稻杂交种花培的影响

采用三种培养基对不同的粳籼稻杂交种F_1进行花药培养。不同的培养基表现出不同的花培效果,基因型之间的培养力差异显著。培养基和基因型之间存在着一定的互作效应。选择培养力高的基因型和与之相适应的培养基是粳籼稻杂交种花培育种的重要因素。     

一个水稻开颖不育突变体ohs1(t)的遗传分析及基因定位

我们在明恢86的转基因后代中发现了一个水稻花器官发育突变体,暂命名为开颖不育(open hull sterile 1,ohs1(t)))突变体.ohs1(t)突变体表现颖花开裂,内外稃片变细,内稃微弯向外稃,有雌雄蕊分化,但雌雄蕊较野生犁株的小,大多数花药没有花粉,少数花药中含有不育花粉,雌雄配子均不育.突变体ohs1(t)分别与明恢86、R527、93-11和中花16号杂交后代遗传分析表明,ohs1(t)是一个隐性基因控制的突变体.以ohs1(t)和93-11杂交F2群体中突变个体作为初步定位群体,采用已报道的SSR标记将OHS1(t)初步定位在1号染色体的长臂端RM493和RM5638两个标记间.随后利用已公布的水稻基因组序列(http://rgp.dna.affrc.go.jp/E/index.html)及93-11和日本晴间SSR标记数据库(http://www.gramene.org/),新开发和筛选了SSR和InDel标记,并以ohs1(t))和中花16号杂交F2群体中突变个体作为新定位群体,将OHS1(t)基因进一步定位在NSSR0115和InDel0102之间,遗传距离分别为0.2 cM和0.3 cM,物理距离约66 kb.