Cytological and molecular biological research on a genic male sterile Chinese cabbage (Brassica campestris L.)

本研究以白菜核雄性不育两用系为材料，采用细胞学观察、组织化学方法对多糖和脂类物质的定位、焦锑酸钾沉淀钙的技术、ATPase-磷酸铅沉淀技术、细胞程序性死亡研究技术以及超微结构观察等方面探讨可育花药和不育花药发育过程中在细胞水平上的差异，并且通过PCR克隆技术克隆得到了白菜核雄性不育性相关的基因片段，同时对该基因片段进行了序列分析及Southern杂交验证。通过比较系统的研究，结论如下：1.通过比较可育株和不育株各个时期花药的结构差异，确定了在减数分裂之前两种花药的母细胞没有明显差异。减数分裂完成以后，不育花药中的小孢子显现出了异常：从四分体中释放出的小孢子外壁不完整。随后不育花药中小孢子的细胞...A male sterile Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) was examined using cytological, cytochemical, calcium-potassium antimonite depositional, ATPase-lead nitrate depositional, and programmed cell death methods to characterize the development of fertile anther and the mechanism of pollen abortion in sterile anther of this plant. Some molecular biological methods were also u...学位：理学博士院系专业：生命科学学院生物学系_植物学学号：B20022600

坛紫菜遗传连锁图谱的构建

以野生型坛紫菜纯系(♀)和红色型坛紫菜纯系(♂)作为杂交亲本,结合四分子分析法及单个体细胞克隆的丝状体途径,创建了由157个株系组成的坛紫菜DH作图群体,并用经过筛选的24对SRAP引物和16对SSR引物对父母本及作图群体各株系进行双标记分析,获得了224个多态性标记,其中157个标记符合孟德尔分离规律。根据标记间的连锁规律,首次构建了坛紫菜的分子遗传连锁图谱,所构建的遗传图谱由包含124个标记(含SRAP标记104个,SSR标记20个)的5个连锁群组成,总长度为879.2cM,平均标记间隔为7.09cM,各个连锁群长度为134.2～213.6cM,包含标记18～31个。最后采用3种不同方法计算得到坛紫菜的估计基因组长度平均为955.3cM,由此得到坛紫菜遗传连锁图谱的基因组覆盖率为92.0%

The Changes in Ca~(2+) level and ultrastructure in the Leaf Cells of Garyota urens L. under Low Temperature Stress

用焦锑酸钙沉淀的电镜细胞化学方法 ,研究了低温胁迫下董棕 (GaryotaurensL .)幼苗叶肉细胞内Ca2 + 水平的变化。研究结果表明 ,未经低温处理的董棕幼苗叶肉细胞 ,焦锑酸钙沉淀颗粒大量出现在液泡和细胞间隙中 ,细胞壁中也可见少量沉淀 ,而细胞基质中则看不到焦锑酸钙沉淀 ;经 2℃ 48h低温处理后 ,细胞基质和细胞膜上焦锑酸钙沉淀增加 ,而液泡和细胞间隙中的焦锑酸钙沉淀则显著减少 ,并且超微结构已初步显示出寒害的特征 ,叶绿体外膜部分破损 ,类囊体片层稀疏且排列不规则 ,光合速率明显下降等 ;经 2℃ 1 2 0h低温处理后 ,细胞间隙内的焦锑酸钙沉淀极少 ,有的也紧贴在细胞外壁上 ,而细胞基质和细胞膜上则分布有非常多的焦锑酸钙沉淀 ,在核基质和液泡中也可见到少量的焦锑酸钙沉淀 ,并且超微结构遭到了显著破坏 ,叶绿体结构完全被破坏 ,核膜与液泡膜严重破损 ,内部结构模糊 ,细胞只表现为呼吸作用 ,不进行光合作用。表明Ca2 + 的区域性分布的变化与植物抗寒性存在一定关系。The changes in Ca 2+ localization in the leaf cells of Garyota urens L. under chilling stress were investigated with calcium antimonate precipitate_electromicroscopic_cytochemical methods. When Garyota urens L. grew on the normal temperature, it was shown that the deposits of calcium antimonate being the indicator for Ca 2+ localization mainly concentrated within the vacuoles and intercellular spaces and there was also some Ca 2+ deposits in cell walls. But when Garyota urens L. was treated by the temperature of 2℃ for 48 h, the level of Ca 2+ increased in cytoplasm and plasma membrane, but decreased in vacuoles and intercellular spaces considerably. At the same time, the ultrastructure of chloroplasts suffered from chilling: the membrane of chloroplasts had been damaged, the layer of thylakoids was exiguous and unclear, the photosynthetic rate decreased evidently. And when Garyota urens L. was treated by the temperature of 2℃ for 120 h, the deposits of Ca 2+ mainly concentrated within the cytoplasm, nucleus and plasma membrane and there was also some Ca 2+ deposits in vacuoles, and the ultrastructure of some cells was simultaneously damaged severely: Chloroplasts structure, vacuole membrane and nuclear membrane had been damaged fully, the structure within the cell had become unclear, and the cell only have respiration. Thus it can be seen that there are some relations between the changes in Ca 2+ distribution within the cell and plant cold_hardiness.国家建设部及厦门市建委资助项

Effects of cold hardening on membrane lipid peroxidation and activities of cell defense enzymes in leaves of Pritchardia gaudichaudii seedling under low temperature stress

在 - 5℃低温胁迫下 ,夏威夷椰子 (PritchardiagaudichaudiiH .Wendl.)幼苗叶片的丙二醛 (MDA)含量逐渐增加 ,表明膜脂过氧化作用逐渐增强 ;含水量不断下降 ;细胞保护酶中的超氧化物歧化酶 (SOD)、过氧化物酶 (POD)和过氧化氢酶 (CAT)酶活性均先升高 ,然后下降。冷锻炼处理可以减缓夏威夷椰子膜脂过氧化作用的增强 ,促进SOD酶活性的提高 ,同时抑制POD和CAT酶活性的变化 ,因而使夏威夷椰子幼苗的抗寒性得以提高 ,在 - 5℃低温胁迫下的半致死时间从 1.6d延长到 2 .2d。Under -5℃ low temperature stress, malondialdehyde (MDA) content in leaves of \%Pritchardia gaudichaudii \%H. Wendl. seedling gradually increased, reflecting that membrane lipid peroxidation aggravated. Water content lowered. Cell defense enzymes SOD (superoxide dimutase), POD (peroxidase) and CAT (catalase) activity raised at early stage, and then lowered. Cold hardening treatment could lessen the increase of membrane lipid peroxidation, promote SOD activity, and inhibite the change of POD and CAT activity under low temperature stress, so the resistance to chilling stress had been enhanced for \%P. gaudichaudii \%seedling. By cold hardening, the half lethal time had been extended to 2.2 d from 1.6 d under -5℃ low temperature stress.国家建设部和厦门市建设委员会资助项

Changes to activated oxygen and protective enzyme acticity in mangrove plant leaf during senescence

测定了红树植物木榄 (Bruguieragymnorrhiza)和秋茄 (Kandeliacandel)幼叶、成熟叶和老叶间活性氧和三种细胞保护酶活性的变化。结果表明 ,在叶片衰老过程中 ,丙二醛和H2O2 含量显著提高 ,超氧化物自由基产生速率加快 ,表明红树叶片衰老中膜脂过氧化作用增强 ,并且与H2O2 和超氧化物自由基含量的上升相关。在3种细胞保护酶中 ,超氧化物歧化酶(SOD)活性逐渐降低 ,过氧化物酶(POD)活性显著提高 ,过氧化氢酶(CAT)活性在叶片成熟过程中增高 ,随着衰老而急剧降低 ,细胞保护酶酶活的变化直接影响了叶片衰老过程中活性氧代谢的平衡。Changes of membrane lipid peroxidation,superoxide free anion generation rate,hydrogen peroxide(H 2 O 2 )content,and activity of some protective enzymes such as superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)were measured in mangrove plant Kandelia candel and Bruguiera gymnorrhiza leaf during senescence.When the leaf grew from the tender stage to the senescence stage,malondialdehyde(MDA)and H 2 O 2 content increased evidently,superoxide free anion generation rate was also gradually raised,showing that membrane lipid peroxidation was enhanced during the leaf growth course,furthermore that itwas related to the raise of H 2 O 2 and superoxide free anion in the leaf.Among these three protective enzymes,SOD activity gradually decreased,while POD activity increased remarkably during growth course.CAT activity increased as the leaf matured,dropping sharply during senescence.Protective enzyme activity in the leaf had an immediate influence on the balance of activated oxygen metabolism.教育部优秀青年教师基金项

Cloning and sequencing of Cu·Zn-superoxide dismutases gene fragment from Avicennia marina

以红树植物白骨壤 (Avicenniamarina)基因组DNA为模板 ,根据超氧化物歧化酶基因保守序列设计特异引物进行PCR扩增 ,得到特异基因片段。回收该基因片段 ,与pMD18 T载体连接 ,并转化到感受态大肠杆菌ER2566细胞 ,获得铜锌超氧化物歧化酶基因片段的克隆。序列分析表明白骨壤铜锌超氧化物歧化酶基因片段含4个外显子和3个内含子 ,编码78个氨基酸,与水稻、玉米、红薯和白杨相应氨基酸序列的同源性分别为83.3% ,84.6% ,84.6%和87.2 %。Special oligonucleotides to highly conserved regions of superoxide dismutases (SOD) gene were used to prime the synthesis and amplification of gene fragment from Avicennia marina genomic DNA by polymerase chain reaction (PCR). The fragment was linked with pMD18 T vector and transformed into the competence cell of Escherichia coli ER2566 strain. After screening of recombinants, detection and sequence analysis, the transformants containing SOD gene were obtained. Sequencing analysis showed that extracted from A. marina Cu·Zn SOD gene fragment,which contains four exons and three introns, had 767 nucleotides. The amino acid sequences which are homologus to those from Cryza sativa, Zea mays, Ipomoea batatas and Populus tremuloid are 83.3%, 84.6%, 84.6% and 87.2% respectively

Changes of calcium ion level in leaf cells from Avicennia marina during leaf senescence

用焦锑酸钙沉淀的电镜细胞化学方法 ,研究了白骨壤叶片衰老过程中叶肉细胞Ca2 + 水平的变化 .结果表明 ,在白骨壤幼叶和成熟叶叶肉细胞中 ,焦锑酸钙沉淀颗粒大量出现在液泡和细胞间隙中 ,细胞壁中也可见少量沉淀 ,而细胞基质中则看不到焦锑酸钙沉淀 .在衰老叶中 ,细胞基质和细胞膜上焦锑酸钙沉淀增加 ,而液泡和细胞间隙中的锑酸钙沉淀则显著减少 ,并且叶绿体外膜部分破损 ,结构破坏 ,核膜与液泡膜内部结构模糊 ,叶绿素含量、净光合速率、气孔导率和蒸腾速率显著下降 .Ca2 + 的区域性分布的变化与植物叶片衰老密切相关Changes of Ca 2+ localization in leaf cell from Avicennia marina during leaf senescence were investigated with calcium antimoniate precipitate electromicroscopic cytochemical methods. In cells of young and matured leaves, the deposits of calcium antimoniate mainly concentrated within the vacuoles and intercellular spaces, and no Ca 2+ deposits existed in cytoplasm. But in cells of old leaves, the level of Ca 2+ increased in cytoplasm and plasma membrane, and decreased considerably in vacuoles and intercellular spaces at the same time. The inner structure and membrane of chloroplasts had been also damaged, nucleus and vacuole membrane were exiguous and unclear, and chlorophyll content decreased in old leaf cell. Conclusively, leaf net photosynthetic rate, stomatal conductance and transpiration rate significantly decreased during senescence. Ca 2+ localization in leaf cell had close relationship with leaf senescence.国家自然科学基金资助项目 ( 4 95 76 2 95

Changes of the Level of Ca~(2+) in Leaf Cells of Pritchardia gaudichaudii H. Wendl. under Low Temperature Stress

用焦锑酸钙沉淀的电镜细胞化学方法 ,研究了低温胁迫下夏威夷椰子 (PritchardiagaudichaudiiH .Wendl.)幼苗叶肉细胞内Ca2 + 水平的变化 .研究结果表明 ,未经低温处理的夏威夷椰子幼苗叶肉细胞 ,焦锑酸钙沉淀颗粒大量出现在液泡中 ,而细胞基质及细胞间隙中则看不到焦锑酸钙沉淀 .而夏威夷椰子幼苗经过 2℃ ,48h低温处理后 ,细胞基质和细胞膜、液泡膜上焦锑酸钙沉淀增加 ,而液泡中的焦锑酸钙沉淀减少 ,并且超微结构已初步显示出寒害的特征 ,叶绿体外膜部分破损 ,类囊体片层稀疏且排列不规则 .而夏威夷椰子幼苗经过 2℃ ,96h低温处理后 ,Ca2 +大量分布于液泡和细胞基质中 ,细胞间隙中也可见少量斑块状沉淀 ,大部分细胞结构已经严重破损 ,内部结构分辨不清呈现出了遭受严重冷害的症状 .从而揭示了Ca2 + 的区域性分布的变化与植物抗寒性的一些关系The changes of Ca 2+ localization in leaf cells of Pritchardia gaudichaudii H. Wendl. under chilling stress were investigated with calcium antimonate precipitate-electromicroscopic-cytochemical methods. When P. gaudichaudii grew at the normal temperature, it was shown that the deposits of calcium antimonate being the indicator for Ca 2+ localization mainly concentrated within the vacuoles. But when P. gaudichaudii was treated at the temperature of 2 ℃ for 48 hours, the level of Ca 2+ increased in cytoplasm and plasma membrane, but decreased in vacuoles considerably. At the same time, the ultrastructure suffered from chilling: the membrane of chloroplasts had been damaged, the layer of thylakoid was exiguous and unclear. And the chilling time was prolonged to 96 hours, the deposits of calcium antimonate mainly concentrated within the cytoplasm and vacuoles and that there was also some Ca 2+ deposits in intercellular spaces, simultaneity, the ultrastructure of some cells was damaged severely: Chloroplasts structure, vacuole membrane and nuclear membrane had been damaged fully, the structure within the cell had become unclear.国家建设部及厦门市建委资助项目 (无编号

THE CYTOCHEMICAL OBSERVATION OF ANTHERS OF CHINESE CABBAGE’S MALE-STERILE

通讯作者：E-mail:[email protected][中文文摘]对一种由一对隐性基因控制的白菜细胞核雄性不育和可育株的花药进行了细胞学和组织化学研究。种子播种后,有1/4植株为不育株,其余的为可育株。通过对不育株和可育株花药发育的细胞学观察,确认不育花粉的败育发生在小孢子发育时期。用组织化学的方法研究了可育株和不育株花药发育过程中的多糖和脂类的分布动态,发现在减数分裂前,可育花药和不育花药的药隔细胞中都储藏了大量的淀粉粒。二者的差异仅是不育花药的绒毡层细胞液泡化明显。在减数分裂后的小孢子发育时期,可育花药的绒毡层细胞具有将药隔细胞中的淀粉粒多糖吸收并转化成脂类的功能,小孢子及以后的二胞花粉中也积累了大量的脂类储藏物质在不育花药中,虽然减数分裂后药隔细胞中的淀粉粒也都消失,但绒毡层细胞中的脂类物质相比很少,同时绒毡层细胞显示了明显的多糖反应,表明不育花药的绒毡层细胞将糖类转化为脂类的功能受阻。在小孢子的表面有些脂类物质,但在细胞质中却没有脂类积累。这一结果暗示在该种白菜细胞核雄性不育株中,由于花药绒毡层细胞转换多糖为脂类的功能失常,导致了小孢子的败育。 [英文文摘]A Chinese cabbage (Brassica campestris L.ssp.chinensis Makino) produces 1/4male sterile and 3/4 fertility in offspring.The sterile plant can be identified from the color ofcorolla that is some white when it grows out.The fertile and sterile anthers were researched us-ing cytological and cytochemical methods.Thick sections of both anthers of different developmen-tal stages were stained with Toluidine blue for general cytological observation and stained withthe periodic-acid-Schiff’s (PAS) technique to detect polysaccharides (red),with Sudan black B(SBB) to detect lipids (black).Before meiosis of microspore mother cells,connective tissue of bothfertile and sterile anthers stored a lot of starch grains.Neither starches nor lipid drops were intapetal trod microspore mother cells.The only difference of both anthers was that the tapetal cellsof sterile anthers contained more vacuoles than those of fertile anthers.After meiosis of mi-crospore mother cells,the starch grains in connective tissue of fertile anthers disappeared,thetapetal cells synthesized abundant lipid drops,and the microspores also began to accumulate lipiddrops.In sterile anthers,the starch grains in connective tissue also disappeared,but only a fewlipid drops appeared in tapetal cells.The tapetal cells,however,became red,suggesting the cellcontained some polysaccharide material.Pollen abortion in sterile anthers occurred in this stage.The aborting microspores accumulated very less lipid drops in its cytoplasm than those in fertileanthers at the same developmental stage.This resuh suggested that in the cabbage,the starchgrains in connective tissue were transformed into polysaccharide and transported to tapetal cells,then these cells transformed polysaccharide into lipid material that was absorbed by developingmicrospore.In sterile anthers,however,polysaccharide in the tapetal cells could not be trans-formed to lipid.The functional default of tapetal cells duriug lipid metabolism led to microsporeabortion.This is new sample in which the functioual default of tapetal cells will make pollenabort,and will enhance research field in male sterile in higher plants

The Electron Microscopic in situ Hybridization and Its Application

将原位分子杂交技术应用于电镜水平 ,观察标记的特定DNA、mRNA和RHA在细胞和 或组织内的超微结构定位的方法 ,称为电镜原位分子杂交技术。本文综述了电镜原位分子杂交技术的建立及其分类 ,并详细介绍了非放射性电镜原位分子杂交技术的基本操作程序及注意事项 ,最后对电镜原位分子杂交技术的应用做了简要介绍。The technique of electron microsco pic \%in situ\% hybridization is applying \%in situ\% hybridization at the electron microscopic level. It is mainly used in the ultrastructural localization of the labled DNA、RNA and RHA in a cell and/or a tissue. In this paper I mainly elaborated its establishment and classification, and the operation procedure of nonradioactive electron microscopic \%in situ \%hybridization and some points for attention. In the end I also discussed its application for research.国家自然科学基金资助 (No .39870 46 1)~