紫稻细胞质雄性不育系叶片全蛋白双向电泳分析

通过对几种不育系叶片全蛋白双向电泳图谱分析证明;紫稻不育系具有不同于野败型,红莲型和马协型不育系的蛋白图谱,说明其可能是一种新的细胞质质源,同时,紫稻不育系与保持系蛋白图谱之间在三叶期和分蘖期时差异均不明显,但各材料本身蛋白图谱在两个不同时期之间差异很大,不育系与保持系图谱表现出的蛋白质（多肽）的差异,可能与不育系雄性不育有关。     

水稻雄性不育系花药微量热分析

采用LKB2277生物活性检测器,对核-质互作雄性不育水稻不育系紫稻A、野败型不育系珍汕97A,红莲型不育系红莲粤泰A和马协型不育系马协A等4个不育系以及1个保持系紫稻B的花药进行了37℃条件下等温呼吸曲线的测定,微量热分析结果表明,每一不育系都具有一特征曲线,等温曲线的幅度高低和走势与花药中花粉粒的败育程度呈睚相关,这与我们的细胞学观察结果相一致。以紫稻A败育最彻底,其呼吸曲线幅度最低,能量代谢最不活跃;余者呼吸曲线幅度递增,依次为珍汕97A,马协A,和红莲粤泰A,最后为紫稻保持系B。     

红莲型和野败型水稻细胞质雄性不育系线粒体DNA(mtDNA)的比较研究

以水稻红莲型和野败型细胞质雄性不育系为材料,用不连续蔗糖梯度离心法提纯线粒体,以饱和酚-氯仿-异戊醇法抽提mtDNA。用琼脂糖电泳和电镜观察比较不育系mtDNA差异,发现不育系中有小分子mtDNA(即m_2和m_3),不同细胞质间的小分子mtDNA存在差异。在不育系与保持系间名为m_1的大分子mtDNA无差异;本文还研究了mtDNAm_1的理化性质。     

水稻野败型细胞质雄性不育系和其保持系蛋白质多肽的比较研究

应用单向SDS—PAGE结合蛋白质铬银染色技术对水稻野败型细胞质雄性不育系珍汕97A和其保持系的叶绿体、线粒体和细胞质的蛋白质多肽进行了比较研究,发现两系之间存在明显的差异,生殖器官(穗子)上的差异比营养器官(叶片)上的差异更为显著。在成熟穗上,叶绿体可溶性蛋白不育系有25条带,保持系仅16条带,两者间有19个多肽不同;线粒体可溶性蛋白不育系有28条带,保持系比不育系少30.1和21.8KD两个多肽;细胞质可溶性蛋白丙酮沉淀物的水溶性蛋白组分不育系有24条带,保持系为29条带,两系间却有7条多肽存在差别;细胞质可溶性蛋白丙酮沉淀物的SDS-增溶性蛋白组分不育系有18条带,保持系只有11条带,两者间亦有7条多肽出现差异。由此可以看出,水稻野败型CMS表型的表达可能需要较多个基因的启动和关闭,既与叶绿体和线粒体有关,还涉及到核基因组的作用。     

五种野生稻叶绿体DNA多态性研究

对野生稻 5个种的18个材料的叶绿体DNA(cpDNA)进行了EcoRI的RFLP分析。 结果显示,共有10种酶切模式,不同种野生稻的cpDNA的RFLP类型都不同,而且在其中一些 种内也有变化,尤以O.rufipogon的种内多态性最为显著,并主要与地理来源有关。本研究还在O.punctata的材料中发现一种以往的分析都不曾描述过的多态性模式。通过对结果的分析,探讨了不同种类野生稻的叶绿体基因组之间以及它们与核基因组之间的进化关系。 Abstract:The polymorphisms of chloroplast DNA from 18 materials of 5 wild rice species were investigated using RFLP analysis.10 restriction patterns were obtained from the analysis of these materials.Different species had different of its RFLP patterns chloroplast DNA,and the polymorphisms existed even with species,especially in O.rufipogon varieties of different geographical origins.In O.punctata a new type of rice chloroplast DNA restriction pattern was discovered which had not been reported before.According to the results obtained,the evolutionary relationships among chlorplast genomes,and between chloroplast and nuclear genomes in different wild rice are discussed.     

水稻线粒体DNA中与雄性不育有关特异片段的克隆及序列分析

该项研究利用RAPD技术,对野败型,矮败型和BT型细胞质雄性不育系,保持系,基因组DNA进行PCR扩增,分离到6条不育系特有的扩增片段,并对野败型,矮败型不育系共有的片段进行了Southern分析,DNA序列分析和SCAR验证,该片段全长1879bp,包含6个开放阅读框架和8对重复序列,BLAST分析表明,该片段部分区域与Elytrigia elongata,小麦线粒体tRNA-Asp基因上游一段序列同源,并对该片段在线粒体DNA中的可能位置等进行了讨论。     

Characterization of Interspecific Hybrids Between Oryza sativa L. and Three Wild Rice Species of China by Genomic In Situ Hybridization

In the genus Oryza, interspecific hybrids are useful bridges for transferring the desired genes from wild species to cultivated rice （Oryza sativa L.）. In the present study, hybrids between O. sativa （AA genome） and three Chinese wild rices, namely O. rufipogon （AA genome）, O. officinalis （CC genome）, and O. meyeriana （GG genome）, were produced. Agricultural traits of the F1 hybrids surveyed were intermediate between their parents and appreciably resembled wild rice parents. Except for the O. sativa × O. rufipogon hybrid, the other F1 hybrids were completely sterile. Genomic in situ hybridization （GISH） was used for hybrid verification. Wild rice genomic DNAs were used as probes and cultivated rice DNA was used as a block. With the exception of O. rufipogon chromosomes, this method distinguished the other two wild rice and cultivated rice chromosomes at the stage of mitotic metaphase with different blocking ratios. The results suggest that a more distant phylogenetic relationship exists between O. meyeriana and O. sativa and that O. rufipogon and O. sativa share a high degree of sequence homology. The average mitotic chromosome length of O. officinalis and O. meyeriana was 1.25- and 1.51-fold that of O. sativa, respectively. 4＇,6＇-Diamidino- 2-phenylindole staining showed that the chromosomes of O. officinalis and O. meyeriana harbored more heterochromatin, suggesting that the C and G genomes were amplified with repetitive sequences compared with the A genome. Although chromocenters formed by chromatin compaction were detected with wild rice-specific signals corresponding to the C and G genomes in discrete domains of the F1 hybrid interphase nuclei, the size and number of O. meyeriana chromocenters were bigger and greater than those of O. officinalis. The present results provide an important understanding of the genomic relationships and a tool for the transfer of useful genes from three native wild rice species in China to cultivars.     

小麦K型及V型细胞质雄性不育系线粒体DNA的比较分析

采用RFLP（restrictionfragmentlengthpolymorphism）和RAPD（radomlyamplifiedpolymorphicDNA）技术对具有相同核遗传背景的小麦K型不育系K149A、V型不育系V149A及相应保持系149B的线粒体DNA进行了比较分析。结果表明它们之间线粒体DNA的结构显著不同,atpA,atp9,coxII,cob等线粒体功能基因有组织结构上的差异。推测线粒体DNA是小麦K、V型雄性不育系胞质不育困子的载体。但由于不育系与保持系间线粒体DNA差异甚大,难以确定究竞哪些差异确与雄性不育有关。仅仅比较不育系和保持系间线粒体DNA的差异是限制本领域取得突破性进展的症结所在。基于“核质工作”的思路,把“雄性不育-育性恢复”作为一个整体来考虑,并重视比较不育系和杂种F1代之间线粒体DNA的结构和表达的差异,所得结果对寻找和鉴定cms因子,进而阐明cms的形成机理有积极意义。     

红莲型杂交水稻的研究与发展

1972年,武汉大学科研人员以红芒野生稻为母本,与江西地方品种莲塘早父本杂交,在后代中发现了雄性不育株.再用莲塘早多代回交,于1974年获得了不育性稳定的株系,命名为红莲型细胞质雄性不育系(Honglian-type cytoplasmic male sterility,HL-CMS),莲塘早为其保持系,红莲型细胞质雄性不育水稻由此问世.与单核期花粉败育的野败型水稻不同,红莲型属于二核期配子败育类型,花粉碘染为圆败,是杂交水稻新质源.40年来,武汉大学科研人员将基础研究、应用研究和产业化紧密结合,促进了红莲型杂交水稻的发展.分子功能验证发现,不育系线粒体内一编码79个氨基酸的可读框OrfH79为HL-CMS不育基因,ORFH79蛋白破坏线粒体正常功能后导致花粉败育.遗传分析发现,HL-CMS为双基因恢复模式,进而克隆了恢复基因Rf5和Rf6.育性恢复机理研究表明,恢复基因RF5蛋白与GRP蛋白互作,组装成400~500kD的分子复合体作用于atp6-OrfH79 mRNA,从而恢复不育性.借助分子标记辅助选择技术,创制出新的CMS和多基因恢复水稻材料,并用于育种实践,选育出高产优质红莲型杂交水稻新组合红莲优6号、珞优8号和粤优9号等,相继通过了湖北省、广西省和农业部国家农作物品种审定委员会等审(认)定,构建的红莲型杂交水稻亲本繁殖提纯体系突破了产业化瓶颈.当前,红莲型杂交水稻在中国长江流域、华南稻区已大面积种植,在越南、印度尼西亚、马来西亚、孟加拉和菲律宾等东南亚国家也表现优异,展现出广阔的应用前景.     

红莲型杂交稻(红莲2号)及其骨干亲本的RAPD分析与鉴定

利用RAPD技术,从248个随机寡核苷酸引物（10－mer）中筛出18个引物对红莲型杂交稻组合红莲2号及其亲本（T－07A、T－07B、YD6－05）,另6个红莲型胞质不育系的骨干恢复和汕优63及其亲本共14份水稻材料进行分析。共检测到173个多态性标记。聚类分析结果表明：不育系与保持系间因核背景相似,遗传差异很小;杂种（F1）的基因型更倾向于恢复系;恢复系与保持系间遗传距离的相对较大,但各恢复系之间的遗传距离较小。利用这些标记能有效地地区交组合中不育系,保持系、恢复系和杂种（F1）。     

Plant regeneration from protoplasts of Indica type rice and CMS rice

Cell suspensions were established from 12 rice genotypes including Indica type, Japonica type and cytoplasmic male sterile (cms) rice lines of both types. 133 green plants were regenerated from protoplasts isolated from an Indica type rice and 15 plants were obtained from a Japonica type cms rice. A cell suspension, from which protoplasts can be isolated (about 10⁶/ml) but from which cell division has not yet been induced, has been established from the most widely used Indica type cms rice which contains Wild Abortive (WA) cytoplasm.     

红莲型细胞质雄性不育水稻线粒体DNA的AP-PCR分析

为了研究红莲型细胞质雄性不育与线粒体基因组的关系。以水稻红莲型粤泰细胞质雄性不育系A和保持系B及杂种一代F1为材料。应用AP-PCR分析,用10个单引物对其线粒体DNA进行扩增。实验结果表明,不同的引物在3种材料间均有不同程度的差异。为红莲型细胞质雄性不育分子机理的研究提供了线索;此外,在引物6F1的扩增图谱中找到一条在YTA和F1中特异的带TAF6F2,Sounthern分析TAF6F2不育胞质的特异性,可能与红莲型水稻细胞质雄性 不育性状的形成有关。     

BT型细胞质雄性不育水稻及其三系的线粒体DNA研究

用RAPD技术对BT型水稻胞质雄性不育系秀A及其保持系秀B、恢复系湘晴以及杂种F1代的线粒体DNA进行了比较分析。结果表明不育系与其保持系间存在显著差异;不育系与其F1之间mtDNA也存在差异。在引物OPJ－08的扩增产物中,秀A扩增出一条分子量为800bp的多态性片段,在引物OPK－10的扩增产物中,杂种F1扩增出一条分子量为900bp的片段。把这两片段回收、克隆并制备探针,OPJ－08800的Southern杂交结果显示不育系与其F1杂交图谱存在多态性;OPK－10900的Suthern杂交结果显示不育系与其保持系同存在差异。推测这两片段与育性可能有一定的联系。     

RFLP Analysis for Mitochondrial Genome of CMS-Rice

Restriction fragment length polymorphism (RFLP) was used to analyze mitochondrial (mt) genome of cytoplasmic male sterility (CMS) rice. Differences were observed among mitochondrial genomes of the sterile line (A) and maintain line (B) of nine types of CMS rice; Mitochondrial genomic differences were also detected between A and B in many functional gene regions. Even the materials with the same nucleic background have differences in their mtDNA. This provides molecular evidence for the cytoplasmic heterogeneity and the CMS mechanism research.     

Alterations of Mitochondrial Protein Assembly and Jasmonic Acid Biosynthesis Pathway in Honglian (HL)-type Cytoplasmic Male Sterility Rice

It has been suggested that the mitochondrial chimeric gene orfH79 is the cause for abortion of microspores in Honglian cytoplasmic male sterile rice, yet little is known regarding its mechanism of action. In this study, we used a mass spectrometry-based quantitative proteomics strategy to compare the mitochondrial proteome between the sterile line Yuetai A and its fertile near-isogenic line Yuetai B. We discovered a reduced quantity of specific proteins in mitochondrial complexes in Yuetai A compared with Yuetai B, indicating a defect in mitochondrial complex assembly in the sterile line. Western blotting showed that ORFH79 protein and ATP1 protein, an F₁ sector component of complex V, are both associated with large protein complexes of similar size. Respiratory complex activity assays and transmission electron microscopy revealed functional and morphological defects in the mitochondria of Yuetai A when compared with Yuetai B. In addition, we identified one sex determination TASSELSEED2-like protein increased in Yuetai A, leading to the discovery of an aberrant variation of the jasmonic acid pathway during the development of microspores.     

细胞质雄性不育水稻线粒体基因组的RFLP分析

利用RFLP技术,比较研究了在农业生产上广泛应用的9种细胞质雄性不育体系的线粒体基因组,结果表明：1）9种水稻雄性不育细胞质的遗传相似性变化范围为0．615～1．000。所有配子体雄性不育细胞质的遗传相似性变化范围为0．6431．000,其中QXA,DY1A和YM15A这3种细胞质的遗传相似性为1．000。所有孢子体雄性不育细胞质的遗传相似性为1．000,2）在配子体细胞质雄性不育水稻中,用3个探针（atpa,atp9＋nad6,cox2）与2种内切酶（HindⅢ,BamHⅠ）的组合未发现不育系与保持系之间的多态性,但在探针atp6,cob,和had2的杂交带型中找到了不育系和保持系之间的一些差异。YTA和YTB在5个探针．内切酶组合（atp61HindⅢ,coblHindⅢ,atp61BamHⅠ,coblBamHⅠ,nad21BamHⅠ）中存在差异。3种细胞质（QX,SJ,DY1）的不育系和保持系之间的差异是相同的,都出现在atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ等3个组合中。YM15A和YM15B在4个组合（atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ,nad2／BamHⅠ）中存在差异。LYA和LYB的差异出现在cob／HindⅢ,cob／BamHⅠ,nad2／BamHⅠ这3个组合中;3）在孢子体细胞质雄性不育水稻中,所有不育系的带型是一样的,所有保持系的带型也一样。不育系和保持系的差异出现在atp6／HindⅢ,cob／HindⅢ,atp6／EcoR,Ⅰcob／EcoRⅠ,cox1／EcoRⅠ,atp6／BamHⅠ,cob／BamHⅠ,cox1／BamHⅠ,cox2／BamHⅠ等9个组合中。这些结果在分子水平上揭示了9种雄性不育细胞质的线粒体基因组存在结构多样性,并为其细胞质雄性不育分子机理的研究打下了基础。     

Damage of oxidative stress on mitochondria during microspores development in Honglian CMS line of rice

One of the cytoplasmic male sterility (CMS) types used for hybrid rice (Oryza sativa L.) production in China is the Honglian (HL)-CMS. Previous studies suggested that pollen abortion of the sterile plants was resulted from a special programmed cell death (PCD) program started at meiosis in the microspores. To elucidate the molecular basis of the pollen abortion, we compared the biochemical and physiological properties such as content of reactive oxygen species (ROS), ATP, NADH, total glutathione and ascorbate acid, the activities of dehydroascrbate reductase, glutathione reductase, ascorbate peroxides and superoxide dismutase, and the integrity of mitochondrial genome DNA isolated from an HL-CMS line, Yuetai A and its maintainer line, Yuetai B. Our results indicated that the mitochondria of the HL-CMS line suffered from a serious oxidative stress during microspores development. Oxidative stress induced by abnormal increased ROS at meiosis stage resulted in the depletion of ATP and NADH, and the degradation of mitochondrial genomic DNA. This suggests that the presence of redox signal originated in mitochondria affects the rest of the cell. Therefore, it is possible that the abortion of premature microspores in HL-CMS line is induced by the chronic oxidative stress in mitochondria in the early stage of pollen development.