An <i>in silico</i>Analysis of Upstream Regulatory Modules (URMs) of Tapetum Specific Genes to Identify Regulatory <i>cis</i>-Elements and Transcription Factors

The present work presents an iin silicoi analysis of Upstream Regulatory Modules (URMs) of genes expressed in tapetum specific manner in dicotyledon and monocotyledon plants. In the current analysis, we identified several motifs conserved in these URMs of which ten were observed to be part of known icisi-elements using tools and databases like MEME, PLACE, MAST and TFSEARCH. We also identified that binding sites for two transcription factors, DOF and WRKY71 were found to be present in majority of the URMs.

Tapetum Degeneration Retardation is Critical for Aliphatic Metabolism and Gene Regulation during Rice Pollen Development

As a complex wall system in flowering plants, the pollen outer wall mainly contains aliphatic sporopollenin; however, the mechanism for synthesizing these lipidic precursors during pollen development remains less well understood. Here, we report on the function of the rice tapetum-expressing TDR （Tapetum Degeneration Retardation） gene in aliphatic metabolism and its regulatory role during rice pollen development. The observations of transmission electron microscopy （TEM） and scanning electron microscopy （SEM） analyses suggested that pollen wall formation was significantly altered in the tdr mutant. The contents of aliphatic compositions of anther were greatly changed in the tdr mutant revealed by GC-MS （gas chromatography-mass spectrometry） testing, particularly less accumulated in fatty acids, primary alcohols, alkanes and alkenes, and an abnormal increase in secondary alcohols with carbon lengths from C29 to C3S in tdr. Microarray data revealed that a group of genes putatively involved in lipid transport and metabolism were significantly altered in the tdr mutant, indicating the critical role of TDR in the formation of the pollen wall. Also, a wide range of genes （236 in total--154 up-regulated and 82 down-regulated） exhibited statistically significant expressional differences between wild-type and tdr. In addition to its function in promoting tapetum PCD, TDR possibly plays crucial regulatory roles in several basic biological processes during rice pollen development.

A Genetic Pathway for Tapetum Development and Function in Arabidopsis

In anther development, tapetal cells take part in complex processes, including endomitosis and apoptosis （programmed cell death）. The tapetum provides many of the proteins, lipids, polysaccharides and other molecules necessary for pollen development. Several transcription factors, including DYT1, TDF1, AMS, MS188 and MS1, have been reported to be essential for tapetum development and function in Arabidopsis thaliana. Here, we present a detailed cytological analysis of knockout mutants for these genes, along with an in situ RNA hybridization experiment and double mutant analysis showing that these transcription factors form a genetic pathway in tapetum development. DYT1, TDF1 and AMS function in early tapetum development, while MS188 and MS1 are important for late tapetum development. The genetic pathway revealed in this work facilitates further investigation of the function and molecular mechanisms of tapetum development in Arabidopsis.

Glycerol-3-Phosphate Acyltransferase 6 （GPAT6） Is Important for Tapetum Development in Arabidopsis and Plays Multiple Roles in Plant Fertility

Glycerol-3-phosphate acyltransferase （GPAT） mediates the initial synthetic step for the formation of glycer- olipids, which act as the major components of biological membranes and the principal stored forms of energy. GPAT6 is a member of the Arabidopsis GPAT family, which is crucial for cutin biosynthesis in sepals and petals. In this work, a func- tional analysis of GPAT6 in anther development and plant fertility was performed. GPAT6 was highly expressed in the tapetum and microspores during anther development. The knockout mutant, gpat6, caused a massive reduction in seed production. This report shows that the ablation of GPAT6 caused defective tapetum development with reduced endoplas- mic reticulum （ER） profiles in the tapetum, which largely led to the abortion of pollen grains and defective pollen wall formation. In addition, pollen germination and pollen tube elongation were affected in the mutant plants. Furthermore, the double mutant analysis showed that GPAT6 and GPAT1 make joint effects on the release of microspores from tetrads and stamen filament elongation. This work shows that GPAT6 plays multiple roles in stamen development and fertility in Arabidopsis.

Premature Tapetum Degeneration: a Major Cause of Abortive Pollen Development in Photoperiod Sensitive Genic Male Sterility in Rice

Photoperiod-sensitive genic male-sterile （PSGMS） rice （Oryza sativa L.）, a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell （PMC） stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL （TdT-mediated dUTP Nick End Labeling） assay, which detects DNA fragmentation resulting from programmed cell death （PCD）, indicating that the premature tapetum degeneration is in the process of PCD.

Cytological and Molecular Characteristics of Pollen Abortion in Lily with Dysplastic Tapetum

Lily was grown worldwide as a fresh cutting flower because of its colorful petals, but its anther contained a large number of pollen grains that cause serious pollen contamination, however, pollen abortion can effectively reduce the level of pollen pollution. Our analysis aims to use cytological observation to detect the critical stage when pollen abortion occurs and to provide comprehensive gene expression information at the transcriptional level. The result showed that pollen abortion in ‘Little Kiss’ began at the mononuclear stage and the callose that covers the microspores failed to degenerate when young pollens were released from the tetrads. In addition, compared with the normally developed one,the tapetum of ‘Little Kiss’ degraded in advance while the degradation of callose was delayed. Furthermore, 103 differentially expressed genes(DEGs) related to the advance degeneration of tapetum cells and callose were found in the expression levels, including 22 transcription factors(TFs). In particular, two β-glucanase genes(endo-1,3(4)-β-glucanase, exo-β-glucanase) responsible for callose degeneration were significantly down-regulated. These results suggested that pollen abortion may occur at mononuclear stage and that early degeneration of tapetum cells resulted in a significant down-regulation of β-glucanase genes. As a result, the callose to cover microspores impedes the formation of pollen walls, which may possibly lead to pollen abortion.

Effects of specific expression of iaaL gene in tobacco tapetum on pollen embryogenesis

The indoleacetic-acid-lysine synthetase (iaaL) gene from Pseudomonas syringae subsp. savastanoi was fused to tobacco tapetum-specific expression promoter TA29, and introduced into tobacco. The expression pattern of this chimeric gene was studied, and the endogenous indoleacetic acid (IAA) levels in different organs were assayed. The results demonstrated that TA29 promoter was only able to direct the specific expression of iaaL gene in transgenic tobacco anther, and resulted in the decrease of endogenous IAA levels in transgenic tobacco anther. No significant phe-notype variation was observed among the transgenic plants at the whole plant level. However, the percentage of pollen embryogenesis was reduced to 11 % when anthers of the transgenic plants were cultured on the modified hormone-free Nistch H (NH) medium, while those of both CK1 and CK2 (see sec. 1.2.2) were more than 50% ; when the an-thers were cultured on NH medium supplemented with 0. 2 mg/L IAA, the percentage of pollen embryogenesis

OsMYB103 is required for rice anther development by regulating tapetum development and exine formation

The Arabidopsis AtMYB103 gene is required for anther development,but whether the homologous gene in rice has the same role is unclear.Sequence analysis indicated that the rice OsMYB103 gene shares a high sequence similarity with AtMYB103.Therefore,we investigated the functional role of OsMYB103 in anther development using an RNAi approach.The OsMYB103 RNA transcript was expressed most abundantly in flowers,specifically in the tapetum,premeiotic pollen mother cells,and meiotic PMCs.OsMYB103-RNAi transgenic lines grew normally during their vegetative phase but displayed reduced male fertility,a phenotype that was associated with downregulated OsMYB103 transcript levels.Expression of OsMS2,an ortholog of the Arabidopsis AtMS2 gene,was also dramatically reduced in the transgenic plants.Knockdown of OsMYB103 led to defects in tapetum development,and most of the microspores in mature anthers lacked exines.Moreover,OsMYB103 could partially rescue the male sterility phenotype of an AtMYB103 knockout mutant ms188.Taken together,these results indicate that OsMYB103 does have an important role in rice tapetum and microspore development.

蕨类植物紫萁绒毡层细胞凋亡的细胞学特征(英文)

绒毡层凋亡过程是小孢子发生中的重要事件,以往的研究主要集中在被子植物,蕨类植物尚未见此方面的报道。该研究首次采用透射电镜和免疫荧光技术对蕨类植物紫萁(Osmunda japonica Thunb.)绒毡层细胞凋亡的细胞学过程进行了观察,以明确紫萁绒毡层细胞的发育类型和凋亡特征,为蕨类植物绒毡层细胞凋亡的深入研究以及孢子发育研究提供依据。结果显示:(1)紫萁的绒毡层属于复合型,即外层绒毡层为分泌型,该层细胞发育过程中液泡化,营养物质被吸收;内层绒毡层为原生质团型,经历了细胞凋亡的过程。(2)绒毡层内层细胞在凋亡过程中细胞壁和细胞膜降解,细胞质浓缩且空泡化;细胞核内陷、变形,染色质浓缩凝聚,形成多数小核仁,DAPI荧光由强变弱;线粒体、质体、内质网、高尔基体等细胞器逐渐退化,液泡中多包含纤维状物、絮状物、黑色嗜锇颗粒和小囊泡等;出现多泡体、多膜体和细胞质凋亡小体,上述特征与种子植物绒毡层凋亡特征基本一致。(3)与种子植物相比,紫萁绒毡层的细胞凋亡开始得早,在整个凋亡过程中没有核凋亡小体的产生;除了产生孢粉素外,绒毡层细胞内产生了大量的丝状物质、絮状物质和电子染色暗的颗粒物,这些物质可能用于形成蕨类植物的孢子周壁。

大葱雄性不育花药败育的形态学特征和细胞学研究

为明确大葱雄性不育株的花器官形态、花药和花粉败育时期及其细胞学机理,采用花器官形态观测、花粉活力检测、石蜡切片、扫描电镜和透射电镜分析等方法,对出口保鲜大葱雄性不育系FCY-A及其保持系FCY-B的花药发育过程和细胞学特征进行观察。结果显示:大葱保持系FCY-B花药饱满,正常开裂散出花粉,花粉活力较强;单个花粉粒呈超长球形,具单沟,外壁纹饰呈脑纹状、较粗糙,表面遍布深浅不一的微穴。而不育系FCY-A花药干瘪,表面未见花粉,花粉无活力;花粉粒间粘连、重叠,未见正常的单粒花粉粒,大多数花粉粒塌陷、变形,表面杂质较多、不光滑且有疣状突起。不育系FCY-A败育时期始于四分体时期,败育原因是绒毡层细胞异常增大,细胞程序化死亡延迟,不能为小孢子发育提供重要的营养物质,导致小孢子空泡化和小孢子外壁发育异常,花粉败育。

通过遗传转化Rfo获得青花菜Ogura CMS恢复系

【目的】将来自萝卜的胞质不育恢复基因Rfo导入到青花菜Ogura细胞质雄性不育(cytoplasmic male sterility,CMS)主栽商业品种‘耐寒优秀’(命名为SFB45)中,获得Ogura CMS恢复系,打破Ogura CMS材料在生产上无法再利用的现状,为优异种质的创制和改良提供新途径。【方法】利用基因合成的方法克隆萝卜Rfo的CDS及其启动子序列,构建表达载体pRfo::Rfo。通过农杆菌介导的遗传转化方法侵染SFB45的具柄子叶和下胚轴,进行再生、抗性苗筛选和PCR鉴定获得阳性植株,开花期观察转基因植株的育性恢复情况;用亚历山大染液分析对照组及阳性株的花粉活力;分别提取对照组及阳性株<3 mm和>3 mm花蕾的RNA并反转录成单链cDNA,设计特异引物,利用RT-PCR分析Rfo及绒毡层和花粉壁发育相关基因在SFB45及对应的育性恢复材料花蕾中的表达差异。【结果】通过遗传转化共获得10个阳性株系,其中8个株系育性得到不同程度的恢复,花粉活力为84.2%—90.4%。RT-PCR分析结果表明,育性恢复植株花蕾中Rfo表达,绒毡层发育关键调节因子DYT1和TDF1及花粉壁主要成分孢粉素合成所必需的基因ACOS5在育性恢复植株<3 mm花蕾中上调表达。绒毡层降解相关基因AMS、四分体胼胝质壁及花粉外壁发育所必需的基因CalS5和CYP703在育性恢复植株>3 mm花蕾中上调表达。分析阳性株系R-1、R-3和R-6自交及杂交后代发现转入的Rfo能稳定遗传,符合孟德尔遗传。【结论】通过遗传转化Rfo获得了优良青花菜Ogura CMS商品种SFB45的育性恢复系,且Rfo的转入使绒毡层及花粉壁发育相关基因恢复正常表达。

高温诱导银灰杨花粉败育的细胞学机理研究

【目的】研究高温处理对银灰杨小孢子发生的影响,揭示高温处理导致杨树花粉败育的细胞学机理,旨在完善高温处理诱导配子染色体加倍、选育林木三倍体的技术。【方法】本研究以银灰杨为试验材料,利用38℃和41℃的高温,对不同减数分裂时期的银灰杨花粉母细胞进行3、6 h处理,研究处理温度、减数分裂时期以及持续处理时间对败育花粉比率的影响。在此基础上,通过比较未经高温处理和高温处理后银灰杨花粉母细胞减数分裂染色体行为、微管骨架动态变化以及减数后绒毡层细胞发育的差异,揭示高温处理诱导银灰杨花粉败育的细胞学机理。【结果】(1)减数分裂时期、处理温度、持续处理时间以及减数分裂时期与处理温度、减数分裂时期与持续处理时间的交互作用均对败育花粉比率具有极显著的影响。41℃高温于中期Ⅰ持续处理3 h的败育花粉比率最高,为(25.11±4.28)%。(2)与对照组相比,银灰杨雄花芽经高温处理后,花粉母细胞减数分裂微管骨架表现出不同程度的解聚,中期Ⅰ和中期Ⅱ的部分纺锤体微管缺失,致使后期Ⅰ和后期Ⅱ的同源染色体或姊妹染色单体分离异常,产生大量的落后染色体。这些落后染色体被遗弃在细胞质中,引起微核的产生,四分体时期形成多分体而导致花粉败育。(3)高温处理可导致银灰杨花药绒毡层退化延迟,但仍能正常开裂,释放出花粉粒,因此绒毡层延迟退化不是高温处理诱导银灰杨花粉败育的原因。【结论】高温处理诱导银灰杨花粉母细胞中期Ⅰ和中期Ⅱ的部分纺锤体微管缺失,致使后期Ⅰ和后期Ⅱ产生大量的落后染色体,引起大量多分体的产生,是高温处理诱导银灰杨花粉败育的细胞学机理。

小麦雄性不育系绒毡层异常代谢与小孢子败育的关系

【目的】研究小麦遗传型雄性不育系花药绒毡层降解及营养物质代谢,并揭示其与花粉败育的关系。【方法】以小麦S型1376不育系[(S)-1376(A)]及其保持系[(A)-1376(B)]为试材,在三核期分别用DAPI和KI-I2对花粉粒内淀粉积累进行染色观察;采用半薄切片技术对不育系(S)-1376和保持系1376绒毡层发育过程及多糖、脂类和蛋白等物质积累进行观察和比较;采用软件cellSens Entry和IPP 6.0分别计算图片中小孢子和绒毡层细胞的面积及分析各发育时期图像中的平均光密度值。【结果】与保持系1376比较,(S)-1376花粉粒被KI-I2染成浅黄色,表明三核期其花粉中无淀粉积累,花粉败育彻底;不育系(S)-1376绒毡层较保持系1376绒毡层细胞提前至四分体时期启动细胞程序化死亡(PCD)过程;不育系(S)-1376花粉核发育迟缓,大多发育至单核晚期停止分裂,仅少数可发育至二核期;(S)-1376小孢子在四分体时期和单核早期显著增大;绒毡层细胞在四分体时期也显著大于保持系1376绒毡层细胞。在(S)-1376花粉的发育过程中,除四分体时期外,其余各时期多糖含量(呈红色)均低于保持系对应的各发育时期,不育系(S)-1376绒毡层中多糖在四分体时期多于保持系,在单核早期显著降低。在花粉整个发育过程中,不育系(S)-1376花粉中被染成黑色脂类的含量明显低于保持系,不育系绒毡层中的脂类含量在各个时期也低于保持系。不育系(S)-1376小孢子母细胞四分体时期被染成蓝色的蛋白含量很高,但单核晚期显著降低;在绒毡层细胞中单核早期蛋白含量显著低于保持系,这可能暗示该时期保持系绒毡层开始启动降解。在花粉发育整个过程中保持系1376小孢子母细胞中四分体时期多糖和蛋白的含量都低于不育系(S)-1376,这可能与该时期不育系绒毡层提前降解需要大量的多糖和蛋白有关;单核晚期小孢子中的多糖、脂类和蛋白含量高于不育系,该时期是正常小孢子发育的关键时期,不育系中各种物质供应不足可能是导致花粉败育的主要原因。在二核期和三核期,不育系(S)-1376花粉营养细胞中大液泡不消失,细胞质内含物也不持续增加,淀粉粒、脂类等物质积累终止,最终导致小孢子内没有内含物的积累,呈空壳晶体状。【结论】小麦S型1376A不育系花药绒毡层提前降解所诱发的物质异常代谢,使小孢子在由单核晚期向二核期发育的过程中物质供应不足导致细胞核分裂异常,最终引起花粉败育。

化学杂交剂诱导油菜雄性不育机理的研究——Ⅰ.杀雄剂1号对甘蓝型油菜花药毡绒层和花粉粒形成的影响

研究揭示了甘蓝型油菜花药壁发育类型属双子叶型,毡绒层为同型单起源毡绒层,又可称为分泌型腺质毡绒层。用0.03％杀雄剂1号处理9个不同发育时期的甘蓝型油莱,研究其诱导雄性不育的效果,结果表明:造胞细胞时期以前处理都是无效的,花粉母细胞以后处理才有效,尤以单核期处理效果最好,不育株率接近100％。这是由于杀雄剂1号诱导甘蓝型油菜花药毡绒层偏离了正常的发育方式,而形成异常的毡绒层,表现为毡绒层增厚,提早解体,与药壁中层分离,并由此产生各种形状的败育花粉。花粉中出现细胞质收缩、空秕,无花粉壁和药中无花粉等现象。文中还对杀雄剂1号诱导甘蓝型油菜雄性不育的作用机制进行了讨论。

辣椒大、小孢子发生与雌、雄配子体发育的研究

运用常规石蜡制片技术与光学显微镜技术研究了辣椒(Capsicumannuum L.)大、小孢子发生以及雌、雄配子体的发育。结果表明:五枚花药,花药四室,花药壁由表皮、药室内壁、2～3层中层和腺质异型绒毡层组成。药室外侧绒毡层由初生周缘细胞衍生而来;药室内侧绒毡层由药隔细胞衍生而来,共同对雄配子体的发育起关键作用。雄蕊中为多孢原,每个药室的横切面为两排小孢子母细胞,经减数分裂后,胞质分裂为同时型,四分体的排列为四面体型和十字型。成熟花粉具3个萌发孔,为二细胞型花粉。中央特立胎座,多胚珠,倒生,单珠被,薄珠心,具有珠被绒毡层。胚珠内为单孢原,孢原直接发育为大孢子母细胞,经减数分裂形成线形四分体,合点端倒数第2个大孢子发育为功能大孢子,经连续三次有丝分裂发育为七细胞七核的成熟胚囊,雌配子体的发育为蓼型。雄蕊发育早于雌蕊,花蕾开放前,雌、雄蕊发育趋于同步。开花时,散出的花粉落到自身雌蕊柱头上,从而实现自花授粉受精。讨论了异型绒毡层的来源、形态结构特点与雄性不育的相关性。

小麦生理型雄性不育花药绒毡层和孢粉素变化与RAFTIN1表达的关系

【目的】研究小麦生理型雄性不育花药绒毡层变化、孢粉素累积与RAFTIN1表达间的关系,为揭示小麦生理型雄性不育的机理奠定基础。【方法】以杀雄剂SQ-1诱导的生理型雄性不育系、质核互作遗传型雄性不育系,正常可育近等基因系为试材,通过石蜡切片、细胞荧光染色和荧光定量PCR技术,研究小孢子不同发育时期花药绒毡层的形态变化、孢粉素的累积及RAFTIN1的表达。【结果】单核期生理型不育系花药绒毡层提前降解,分泌孢粉素的含量降低,RAFTIN1提前高表达,使大量孢粉素转运至花粉壁层;二核期和三核期,生理型不育系绒毡层完全退化,停止分泌孢粉素,RAFTIN1呈现明显的下调表达模式。【结论】杀雄剂SQ-1诱导的小麦生理型雄性不育其败育机理与绒毡层的提前降解和定向转运孢粉素的RAFTIN1表达高低直接相关。

棉花晋A细胞质雄性不育系小孢子发生的显微和超微结构观察

研究了棉花晋 A细胞质雄性不育系的细胞学特征。结果表明 ,1晋 A不育系雄性败育的主要时期是在造孢细胞增殖——小孢子母细胞形成时期。大部分小孢子母细胞在形成过程中退化解体 ;少数小孢子母细胞能进一步发育并开始减数分裂 ,但都仅停留在减数分裂前期 ,该不育系绒毡层提早解体与小孢子母细胞解体有关。 2小孢子母细胞和绒毡层细胞中的线粒体异常 ,对晋 A不育系小孢子败育有直接的影响。线粒体异常表现的特征为 :线粒体肿胀破裂 ,内嵴溶解。

白菜OguCMS相关MYB家族新基因BcMYBogu的克隆与特征分析

为研究CMS核质互作的分子机理,将甘蓝型油菜(Brassica napus L.)和白菜(B.campestris L.ssp.chinensis Makino)杂交并连续回交6代获得白菜OguCMS,在与保持系花药细胞学比较的基础上,运用cDNA-AFLP筛选得到白菜OguCMS早、中期花蕾提早表达的MYB-like差异片段,利用RACE克隆得到该片段的cDNA全长,命名为BcMYBogu(GenBank登录号:EF127861),对其氨基酸序列和表达特征进行研究。结果表明,白菜OguCMS绒毡层在四分体后增生,高度液泡化,导致小孢子花粉外壁异常,细胞质同外壁分离并降解;花药变白;BcMYBogu具有典型的MYBDNA结合域—W残基和SH[AL]QKY[RF]基序;系统进化分析显示BcMYBogu与AtMYB26,AtMYB32和AtMYB4等聚类在同一分枝;RT-PCR分析表明BcMYBogu在莲座叶、花茎和花蕾中均有表达,但在OguCMS花蕾中表达量显著上升。由此推测BcMYBogu是一个新的与白菜OguCMS相关的MYB家族新成员。

高粱A_2型细胞质雄性不育系小孢子发生的细胞学观察和减数分裂染色体行为分析

高粱A2型细胞质雄性不育性(CMS)的细胞质来源于IS12662C,A2细胞质杂交种目前已用于生产。本文以A2/B2V4为材料,对A2CMS小孢子败育过程作了细胞学观察,并对小孢子败育过程中减数分裂的染色体行为作了分析。研究发现,在A2雄性不育系A2V4的花药发育过程中,绒毡层细胞不形成或提前解体;绒毡层细胞畸形化;绒毡层细胞虽发育正常,但小孢子母细胞减数分裂行为异常;这些都导致小孢子退化。A2细胞质雄性不育花粉母细胞减数分裂行为从后期Ⅰ开始出现异常,同源或姊妹染色体向两极分离时滞后或不分裂;染色体多倍化;一个细胞内出现多核和多核仁现象,最终导致小孢子败育。

辐射诱变水稻雄性不育突变体tda的遗传与细胞学分析

为了挖掘更多与水稻花药发育相关的基因,利用60Co-γ射线辐照粳稻品种松香早粳,获得1个水稻雄性不育突变体tda,并对其细胞学和遗传学进行分析。结果表明,tda突变体在营养生长期无明显的表型缺陷,花序和花器官能正常发育,但其花药较小且呈白色。组织切片研究发现,tda突变体在小孢子发育时期开始出现异常,绒毡层提前降解,小孢子呈畸形状,随后小孢子萎缩不能形成正常的花粉粒。遗传学分析表明,tda是一个单基因控制的隐性核突变体。该研究结果为TDA基因的克隆、功能分析及水稻花药发育的分子机制奠定了基础。