顺乌头酸酶基因在杀雄剂SQ-1诱导小麦雄性不育花药中的表达分析

采用RT-PCR技术,克隆了小麦胞质顺乌头酸酶基因(cACO)部分cDNA序列.该cDNA序列长1368bp,编码456个氨基酸,GenBank登录号为GU475062.半定量RT-PCR结果表明,在生理型不育和可育花药发育的单核早期至三核期,cACO基因的表达水平均表现为先升后降;在生理型不育花药发育的单核晚期cACO基因表达水平与同期可育花药相比显著升高,到二核期和三核期明显降低,ACO酶活性变化表现出相同趋势.这反映出在小麦生理型不育系中,cACO基因在花药败育关键期异常表达可能影响了花药发育过程中正常的能量供应和物质代谢,导致花粉发育能量不足和所需物质匮乏,从而导致了非遗传型花药败育现象.     

基因芯片分析杀雄剂SQ-1诱导小麦生理型雄性不育的基因差异表达

为揭示小麦生理型雄性不育的分子机理,更好地为小麦杂种优势利用提供理论依据和技术支撑,本研究以SQ-1诱导的西农558生理型雄性不育的花药为试验材料,以未经SQ-1处理的西农558的花药为对照,利用基因芯片技术对两者的基因表达差异进行了分析,并对部分基因运用半定量PCR技术进行了验证.结果表明,在55 052个转录本中, 两材料间差异表达的转录本有2 052条, 其中1 294个基因表达上调,758个基因表达下调.功能分类表明这些基因主要参与了毒性物质响应、逆境响应、多糖代谢及信号转导等重要生命过程.为验证芯片数据的可信性, 利用cDNA半定量 PCR 法对11个差异表达显著的基因(Ta.116, Ta.5629, TaAffx.122333, Ta.30726, Ta.13682, Ta.4057, Ta.4101, Ta.4139, Ta.11957, Ta.25934, Ta.27552) 进行验证.结果证明,无论是上调表达的还是下调表达的基因,其表达模式都与基因芯片的检测结果的一致.这些基因可作为育性相关候选基因开展下一步研究.     

杀雄剂SQ-1诱导小麦雄性不育花粉粒差异蛋白质组学研究

采用固相pH梯度/SDS-PAGE双向凝胶电泳对经杀雄剂SQ-1处理和未处理的小麦(Triticum aestivum)成熟期花粉总蛋白质进行了分离, 银染显色, 获得了分辨率和重复性较好的双向电泳图谱. 通过PDQuest 2DE图像软件的分析, 在等电点4～7之间可识别350个以上较为清晰的蛋白质点, 其中差异表达明显的蛋白质点数为21个. 将11个差异点采用基质辅助激光解析电离飞行时间质谱进行了肽质量指纹图谱分析, 采用Mascot软件在Swiss-prot数据库查询, 鉴定出了7个蛋白质, 它们分别是液泡转化酶、动力蛋白轻链TCTEX-1、锰超氧化物歧化酶、果糖-1,6-二磷酸醛缩酶、抗坏血酸过氧化物酶、凝集素蛋白激酶和一种未知功能的蛋白. 对已知蛋白的功能进行分析, 推测杀雄剂SQ-1诱导小麦雄性不育可能与能量代谢失衡、淀粉合成受抑制、活性氧积累、细胞凋亡以及花器官发育调节基因作用失控等有关.     

化学杂交剂诱导的小麦生理型雄性不育花药的活性氧代谢

以化学杂交剂SQ-1诱导的生理型小麦雄性不育及其对照植株花药为材料,研究了不同花粉发育时期花药中超氧阴离子(O-·2)生成速率、过氧化氢(H2O2)和丙二醛(MDA)含量以及主要抗氧化酶活性的变化,以探明小麦花药活性氧代谢和生理型雄性不育的关系.结果表明,在幼穗时期,O-·2生成速率、H2O2和MDA含量、超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性均高于相应对照,而过氧化物酶(POD)和过氧化氢酶(CAT)活性则低于或显著低于对照;在单核早期以及花粉败育主要发生期(单核后期和二核初期),O-·2生成速率、H2O2和MDA含量极显著高于对照,而SOD、POD、CAT和APX酶活性却极显著低于对照;在败育后的花药中,O-·2生成速率和H2O2含量与对照之间差异幅度缩小,但MDA含量依然加大,同期的几种抗氧化酶活性依然极显著低于对照.在败育的关键期,品种'西农1376'处理株花药的活性氧升高幅度比'西农2611'处理株较大,抗氧化酶活性降低幅度也较大,且'西农1376'处理株的相对雄性不育率也较高.可见,化学杂交剂SQ-1能诱导小麦花药中O-·2和H2O2大量积累以及SOD、POD、CAT和APX活性的极显著降低,引起花粉关键败育期花药活性氧代谢严重失衡和严重膜脂过氧化,导致大量花粉母细胞发育受到严重抑制,最终造成小麦生理型雄性不育.     

Development of Tapet urn and Microspores in Canna L.: an Example of an Invasive but Non-syncytial Tapetum

An investigation of microsporogenesis in Canna L. revealed thatthe tapetum is invasive but non-plasmodial. Tapetal cell protoplastsare released as individuals. In the loculus they are at firstmore or less spherical but can produce amoeboid processes inlate stages of microsporogenesis. They do not fuse with oneanother. Meiosis is normal and cytokinesis is successive. Theimplications of the novel type of tapetum are discussed. canna, Canna, invasive non-syncytial tapetum, tapetum, microsporogenesis     

Cytological and Comparative Proteomic Analyses on Male Sterility in Brassica napus L. Induced by the Chemical Hybridization Agent Monosulphuron Ester Sodium

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indicated that defective tapetal cells and abnormal microspores were gradually generated in the developing anthers of MES-treated plants at various development stages, resulting in unviable microspores and male sterility. A total of 141 differentially expressed proteins between the MES-treated and control plants were revealed, and 131 of them were further identified by MALDI-TOF/TOF MS. Most of these proteins decreased in abundance in tissues of MES-treated rapeseed plants, and only a few increased. Notably, some proteins were absent or induced in developing anthers after MES treatment. These proteins were involved in several processes that may be crucial for tapetum and microspore development. Down-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in defective tapetum and abnormal microspores that lead to male sterility in MES-treated plants. Accordingly, a simple model of CHA-MES-induced male sterility in rapeseed was established. This study is the first cytological and dynamic proteomic investigation on CHA-MES-induced male sterility in rapeseed, and the results provide new insights into the molecular events of male sterility.     

Alterations and Abnormal Mitosis of Wheat Chromosomes Induced by Wheat-Rye Monosomic Addition Lines

Background

Wheat-rye addition lines are an old topic. However, the alterations and abnormal mitotic behaviours of wheat chromosomes caused by wheat-rye monosomic addition lines are seldom reported.

Methodology/Principal Findings

Octoploid triticale was derived from common wheat T. aestivum L. ‘Mianyang11’×rye S. cereale L. ‘Kustro’ and some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ followed by self-fertilization. Genomic in situ hybridization (GISH) using rye genomic DNA and fluorescence in situ hybridization (FISH) using repetitive sequences pAs1 and pSc119.2 as probes were used to analyze the mitotic chromosomes of these progeny. Strong pSc119.2 FISH signals could be observed at the telomeric regions of 3DS arms in ‘Mianyang11’. However, the pSc119.2 FISH signals were disappeared from the selfed progeny of 4R monosomic addition line and the changed 3D chromosomes could be transmitted to next generation stably. In one of the selfed progeny of 7R monosomic addition line, one 2D chromosome was broken and three 4A chromosomes were observed. In the selfed progeny of 6R monosomic addition line, structural variation and abnormal mitotic behaviour of 3D chromosome were detected. Additionally, 1A and 4B chromosomes were eliminated from some of the progeny of 6R monosomic addition line.

Conclusions/Significance

These results indicated that single rye chromosome added to wheat might cause alterations and abnormal mitotic behaviours of wheat chromosomes and it is possible that the stress caused by single alien chromosome might be one of the factors that induced karyotype alteration of wheat.     

用抑制差减杂交法分离小麦幼苗水分胁迫诱导表达的cDNA

小麦种子水培 ,幼苗长至一叶一心后 ,在 2 0℃生长箱中水培 4 8h作为对照组 (Driver) ,PEG 6 0 0 0水溶液胁迫培养 4 8h作为处理组 (Tester)。进行抑制差减杂交 ,构建包含 15 0 0个独立克隆的SSH文库。以正向和反向差减杂交后的cDNA为探针 ,筛选SSH文库 ,得到 181个阳性克隆 ,测序后获不重复EST 10 1个。     

Fertilization and Embryo Development in Hybridization Between Wheat and Leymus

Observations were made of the fertilization and embryo development in intergeneric cross between Triticum aestivum L. cv. Chinese Spring and Leyrnus secalinus Tzrel. The pollen germination of Leymus secalinus appeared normal on the stigma of Triticum aestivum and the pollen tubes grew into the style and entered the embryo sacs. Double and simple fertilization were observed in the pollinated florets. Of the 319 ovaries examed 62 (19.44%) had double fertilization and had embryo and endosperm, but endosperm development was slower than that of the embryos, 49 (15.36%) had only embryo and 7 (2.19%) had only endosperm. The total percentage of fertilization was as high as 36.99%. However, only I seed was obtained from 150 wheat ftorets pollinated with Leymus secalinus. This was obviously due to the absence or poor development of the endosperm. It may be suggested that the potential of increasing the frequency of hybrid plant obtainment was great in the cross between wheat and leymus, if embryo culture technique is employed at the early stage of hybrid embryo development.     

Abnormal Sporogenesis in Wheat (Triticum aestivum L.) Induced by Short Periods of High Temperature

Plants of Triticum aestivum L. cv. Gabo, grown at 20 °C,were exposed to 30 °C for short periods during the timebetween the beginning of meiosis in the pollen mother cellsand anthesis. Plant water deficit at this temperature was avoidedby maintaining a high atmospheric relative humidity and tissuewater potential did not change. This temperature treatment appliedfor 3 days, at the time of reduction division and tetrad breakup in the male tissue, lowered grain yield through a drasticreduction in grain set, but was without effect at other stagesof development. Grain set was also reduced by exposing plantsto 30 °C for 1 day only or to a 30 °C day, 20 °Cnight (16 h photoperiod) regime for 3 days during the sensitiveperiod. A reduction in grain set did not result in a compensatoryincrease in the weight of remaining grains. The female fertility of previously heat-stressed plants wasassessed by pollinating with pollen from plants grown at a lowertemperature (20 °C). Grain set in such plants was less thanthat in plants grown at the lower temperature and hand pollinatedwith similar pollen, indicating that female fertility was reducedby high temperature. This was not the sole reason for reducedgrain set, however, as some anthers on heat-stressed plantswere small and neither extruded nor dehisced normally. Suchanthers contained pollen grains that were mostly shrivelled,had abnormal cytoplasm and showed no reaction to 2, 3, 5-triphenyltetrazolium chloride. Similar effects were also noted in pollenfrom apparently normal anthers on heat-stressed plants. Triticum aestivum, wheat, heat stress, pollen, sporogenesis, grain set, male sterility, female sterility     

小麦x长穗偃麦草的受精和早期胚胎发育

用石蜡切片法,对小麦(Triticum aestivum)和长穗偃麦草(Elytrigia elongata)杂交的受精和早期胚胎发育进行了观察。结果表明,长穗偃麦草花粉在小麦柱头上萌发良好,花粉管可顺利长人花柱和胚囊。 观察的170个小麦子房中,17.65％发生了双受精,产生了胚和胚乳;9.41％发生了单卵受精,只产生胚而无胚乳;4.71％。发生了单极核受精,只产生胚乳而无胚;总受精率为31.77％;成胚率为27.06％。由于胚乳的缺乏或发育异常及败育,最终难以获得有生活力的种子。为小麦与长穗偃麦草远缘杂交提供了细胞胚胎学证据。     

Development of the Tapetum in Pinus banksiana Preceding Sporogenesis

Early in sporangial ontogeny, the cells destined to become thesporogenous and tapetal tissue differentiate in a strikinglysimilar manner. The first conspicuous step in development isa contraction of the protoplasts, beginning at the centre ofthe microsporangium and moving radially to its periphery. Similardevelopment of the two groups of cells ceases as the callosewall is formed around the meiocytes. At this point the originalwalls investing the tapetal cells become gelatinous, and lipidsynthesis commences within the contracted protoplasts. The bulkof this lipid is secreted from the cells, and becomes lodgedin the loculus, either as globules in the expanded radial andinner cell walls, or as a continuous layer on the inside ofthe middle lamella separating the loculus from the wall of themicrosporangium. This lipoidal layer forms the basement of aperitapetal membrane, believed to serve as a container for thefluid in which the young sporogenous cells are immersed. Examination of protein levels and ribosome numbers in the tapetalcells reveals that protein synthesis proceeds at an increasingrate throughout the development preceding meiosis, but apparentlyceases as the pollen mother cells become enveloped in callose.     

Identification of Alien Chromatin and Ribosomal DNA in Wheat by in Situ Hybridization

In situ hybridization was carried out to somatic cells of hexaploid Triticale “Badger”, lB/IR translocation line “Ning 8026” and IR(ID) substitution line “84056-1-36-1” using biotin-labelled total rye genomic DNA and wheat rDNA as probes, the results were as follows: 1. The probe containing the total genomic DNA from rye hybridized to the entire length of all rye chromosomes, as a result of the formation of a brown precipitate over the sites of hybridization, the rye chromosomes could be distinguished from wheat chromosomes counterstained by Wright’s solution, the distinguishable appearance of the wheat and rye chromosomes resulted in an efficient method of detecting rye chromosome or segments in wheat. 2. When the probe PTA 71 containing wheat ribosomal DNA was used to hybridize to somatic chromosomes of "Badger" and “84056-1-36-1”, six signals in “Badger” and eight in “84056-1-36-1” were observed on lB, 6B, 1R and SD, among which lB and 6B showed large in situ signals corresponding to many copies of the genes. 3. The expression behavior of wheat rDNA was found in interphase cells by in situ hybridization.     

利用荧光原位杂交技术检测导入普通小麦的大赖草染色质

利用以大赖草基因组ＤＮＡ为探针的荧光原位杂交技术对导入普通小麦的大赖草染色质进行检测。结果：９１）根尖体中花粉母细胞时期均可用于检测大赖草染色质。间期核中杂交信号点数与所含大赖草染色体数目之间的对应关系依染色体显强Ｃ－带末端数不同而不同;（２）利用荧光原位杂交,从普通小麦－大赖草单体异附加系的减数分裂前植株＾６０Ｃｏ－γ射线辐射后代中检测到一批大赖草端着丝粒染色体和小麦－大赖草染色体易位等结构变异     

Electron-Microscope Observation on the Pollen Development of Male Sterility Wheat Induced by Ethrel

The pollen development of male sterility wheat induced by Ethrel was studied in comparison with that of normal wheat by transmission and scanning electron-microscope. The results obtained are summarized as follows: 1. The primary morphological changes of abortive wheat pollen after treatment with Ethrel took place in the vacuole stage. The materials of cytoplasm were rarefied. All kinds of the cell organelles and vacuoles became degenerated and disorganized. Inside a small number of cell nuclei, chromatin granules coagulated irregularly. The number and the activity of the Ubisch bodices became reduced evidently. 2. In the mature stage, the differences between the treated wheat and normal wheat became even more striking. The normal pollens were spherical in sharp, and full of starch granules. The treated pollen were monstrous. There was a large empty vacuole in every abortive pollen cell, in which the starch granules were smaller and fewer than these in the normal ones. The nuclei and cytoplasm in some of the abortive pollens were degenerated, leaving only the cell walls. 3. Under scanning electron microscope, it was discovered that the normal wheat pollen were spherical or oval. The germinated pores jutted. The round-openings of the pores could be clearly seen. The abortive pollen induced by Ethrel looked like some shrunken balls, and became deformed and were blocked up. 4. On discussion of the mechanism of the Ethrel-induced male sterility in wheat, the authors suggest that special attention should be paid to the changes of the Ubisch bodies and the vacuoles.     

DNA Synthesis in Microspores in Anther Culture of Wheat (Triticum aestivum L.)

Anthers with mid-unlnucleate microspores were cultured on W5 medium supplemented with 0.5 mg/l kinetin, 2 mg/l 2,4-D and 9% or 3% sucrose. At a series of interval (0, 1, 1.5, 2, 14 days) after cultured, the anthers were labelled with 3H-thymidine (4 MCi/mi) for 24 h, fixed, and then performed autoradiography according to conventional method. Results show that after cultured for 24 h, 3H-thymidine was incorporated into some late-uninucleate microspores (see Plate I, 3), and after for 2.5 days, vegetative nuclei in pollen grains were la- belled (see Plate I, 4). Usually, vegetative nuclei were labelled frequently and generative ones were labelled rarely. Sometimes generative cell which could synthesis DNA might develop suspensor-like structure individually (see Plate I, 13). During early stage of development of a multicellular pollen grain, the DNA synthesis in the cells were synchronized. With pollen development, the synchronism of DNA synthesis was destroyed. When anthers cultured on medium with 3% sucrose, DNA in microspores could be synthesized normally, and the number of labelled microspores was more than that of anthers cultured on medium with 9% sucrose. However, on medium with 3% sucrose, the nuclei in microspores stopped dividing after one or two divisions and the cell wall of them could not be formed and multicellular pollen was not observed. It seems that the absence of multicellular pollen on medium with 3% sucrose was primarily due to the block of cell division and cell wall formation, not due to the interruption of DNA synthesis.     

Ocular Lesions Induced by the Trachoma Agent in Rabbits

Injection of the T'ang strain of trachoma agent into the anterior chamber of the rabbit eye caused corneal opacity, characteristic microscopic lesions of the corneal endothelium, varying degrees of neovascularization of the cornea, and uveitis. These ocular changes were agent-specific. The experimental data showed that the microorganism developed in the ocular tissues. The results of this study suggest the possibility of using the rabbit eye as an experimental model for the study of trachoma.