萍乡显性雄性核不育水稻超微结构研究

利用电镜技术对萍乡显性核不育水稻可育株和不育株花粉形成及发育过程和药壁组织的基本结构及其发育进行了研究。导致了其不育花粉败育的主要原因有：（1）绒毡层细胞表现为延迟解体,乌氏体不能与小孢子细胞壁的形成,特别是纯合不育株绒毡层细胞存在乌氏体的异位分布;（2）药隔维管束异常,导致营养物质运输缺乏,薄壁细胞液泡化,排列紊乱,杂合不育株薄壁细胞互相融合,出现核转移的独特现象。     

水稻“三明”显性核不育基因的定位

"三明显性核不育水稻"突变体是由福建省三明市农业科学研究所于2001年在杂交组合"SE21S/Basmati370"的F2代群体中发现的。其不育性受1个显性基因控制(将该基因命名为SMS)。经过多代回交,该显性不育基因已导入籼稻品种佳福占的遗传背景中(将该不育材料称为佳不育)。为了定位SMS,文章将佳不育与粳稻品种日本晴杂交,并将F1与佳福占测交,构建了一个作图群体。利用SSR和INDEL标记,通过混合分离分析和连锁分析,将SMS定位于第8号染色体上两个INDEL标记ZM30和ZM9之间,约99 kb的区间内。该结果为克隆SMS奠定了基础。     

萍乡显性核不育水稻育性相关基因的定位和遗传分析

萍乡显性核不育水稻(Pingxiang Dominant Genic Male Sterile Rice,PDGMSR)是在水稻中首次发现的显性核不育材料,其育性由两对显性基因互作控制,一对是萍乡显性核不育基因Ms-p,另一对是显性上位恢复基因(dominant epistatic fertility restorer gene,Rfe)。两者共同存在时显性上位恢复基因能抑制不育基因的表达,从而使育性表现可育。本实验用一个对萍乡显性核不育水稻有恢复能力的水稻品种E823与萍乡显性核不育水稻配制杂交组合,将(萍乡核不育水稻/E823)F2作为定位群体,根据F3株系的育性分离,选择育性分离株系对应F2单株(基因型为Ms-pMs-pRefrfe和Ms-pms-pRferfe)构建可育池,用对应F2株系中的不育单株(基因型为Ms-pMs-prferfe或Ms-pms-prferfe)构建不育池,将显性上位恢复基因Rfe定位在水稻10染色体RM311和RM3152一侧,遗传距离分别为7.9cM和3.6cM。根据已有的Ms-p的定位结果,合成10染色体部分微卫星引物,对不育单株进行分析,发现RM171和RM6745位于Ms-p的两侧,距离分别为0.3cM和3.0cM。根据10染色体的测序结果,将Ms-p界定在约730kb的范围内,并构建了Ms-p的电子重叠群。植物显性核不育的育性恢复机理存在“复等位基因”和“显性上位互作”两种假说,贺浩华等用经典的遗传学方法证明了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互作”。理论上认为,确定其遗传机理最为有效的方法是基因定位,如果不育基因和恢复基因位于同一位点,则其遗传机理属于“复等位基因”,否则为“显性上位互作”。本实验将不育基因和恢复基因定位在水稻10染色体不同的位点,用基因定位的方法证实了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互作”。     

萍乡显性核不育水稻花粉败育的细胞形态学观察

利用光学显微镜技术对萍乡显性不育水稻（PXDGMSR）可育株和不育株花粉形成及发育过程,药壁组织的基本结构及其发育进行了研究,导致其不育株花粉败育的主要原因有：（1）绒毡层细胞解体延迟;（2）花粉母细胞减数分裂方式为“连续型”,但分裂期细胞液泡化严重,染色体粘连,纺缍体形成不规则,核中出现囊泡化现象,（3）花粉母细胞减数分裂方式为“同时型”,母细胞形成多核现象。     

温敏雄性核不育水稻可育花药与不育花药的钙分布

用焦锑酸盐沉淀法研究了温敏雄性核不育水稻在减数分裂时期和单核早期可育花药与不育花药的钙分布.结果表明：在减数分裂时期,可育花药小孢子母细胞和药室内的钙颗粒很少,而不育花药小孢子母细胞中分布许多的钙颗粒,特别是药室中的钙颗粒异常丰富,小孢子母细胞减数分裂异常,细胞质收缩退化.在单核早期,可育花药花粉内的钙颗粒极少,花粉表面分布许多钙颗粒,而不育花药花粉内分布许多钙颗粒,药室内的钙颗粒仍然非常丰富.可育花药维管束鞘细胞体积大且形状规则,细胞内的钙颗粒很少,而不育花药维管束鞘细胞体积小且形状不规则,细胞内的钙颗粒较多.     

mRNA差异展示研究胃癌差异表达基因

以胃癌细胞株GC7901与胃粘膜细胞株GES-1为对比材料,利用mRNA差异展示技术,研究胃粘膜至胃癌过程的差异表达基因,为建立胃癌预警系统打下基础.获得8个未知的与胃癌相关的cDNA,命名为GCYS-1,2,3,4,5,6,7,20,完成其克隆及序列分析,RNA印迹证实GCYS-1至7片段与GCYS-20基因在GC7901细胞株中呈高表达,在GES-1细胞株中呈低表达.此8个序列在GenBank数据库中登录,接受号为AF054162～AF056168及AF219140.     

大白菜显性核不育基因向白菜型油菜上转育的遗传研究

利用大白菜的显性核不育基因向白菜型油菜上转育,成功地育成了白菜型油菜３种不育系。其配合力测定的总趋势：甲型不育＞全不育＞乙型不育。经遗传分析、选育,找到了上位显性核互作恢复基因（Ｍｓ）,它对核不育基因（Ｓｐ）具有上位恢复作用。育性是由２对显性核不育基因（Ｓｐ－）和上位显性核互作恢复基因（Ｍｓ－）控制的。并弄清了其相应遗传模式。     

水稻雄性不育与可育花药的mRNA差别显示和cDNA差别片段的分析

用mRNA差别显示 ,对水稻细胞质雄性不育系、保持系和F1杂种的花药mRNA和叶片mRNA进行了比较和分析 ,以研究雄性不育花药在花粉败育时期的基因表达方式 .花药在不育与可育间显示的cDNA差别带数多于叶片 ,表明在花药中育性基因的表达比叶片中活跃和充分 .不同类型花药的基因转录方式既与花药育性程度有关也与花药败育早晚有关 ,不育、部分不育和早期败育的花药所显示的cDNA差别带数多于可育和晚期败育的花药 .在回收的 1 2个cDNA差别片段中 ,有2个可能与雄性不育相关 ,AB₄A₅ 片段只在不育花药中专一表达 ,另一片段AB₃ B₂ 含有与线粒体基因coxⅡ同源的序列 ,在不育花药中的表达受到部分抑制     

雄性核不育水稻育性转换的光周期效应指数值(PE)和温度效应指数值(TE)的遗传性初步研究

对比考察了农垦５８ｓ与７００１ｓ、８９０２ｓ与培矮６４ｓ及８９０２ｓ与安农Ｓ１等３个光（温）敏核不育水稻杂交组合的双亲、Ｆ１、Ｂ１、Ｂ２和Ｆ２世代的样本及每一个体的ＰＥ和ＴＥ的变异。无论双亲核不育基因的等位程度及栽培环境的光、温周期如何,在多数情况下都显示了如下的规律：（１）后代样本ＰＥ、ＴＥ的大小虽然形形色色,但都取决于其双亲。Ｆ１表示完全显性甚至超显性。（２）Ｆ２个体的ＰＥ或ＴＥ、尤其同一个体ＰＥ与ＴＥ的集成类型发生有规律的分离和超亲分离,产生形形色色的育性转换类型。在纯粹由雄性不育个体组成的Ｆ２不育分样本中也发生同样分离。（３）ＰＥ或ＴＥ的广义遗传力的估值都大于５０％。据此推断ＰＥ、ＴＥ是两个可遗传并可供选择的独立性状;并对育性转换现象的遗传机制也进行了讨论。     

差异表达基因分离技术的研究进展

分离并克隆差异表达基因是生命科学的研究热点.近年来,以差示筛选、扣除杂交等基本方法为基础,先后出现了抑制差减杂交,微阵列技术等多种分析差异表达基因的技术, 使差异表达基因分离方法不断完善.对这些方法的优缺点、发展趋势及应用前景进行了简要综述.     

亚麻中雄性不育基因同源序列MS2-F的克隆和表达分析

用同源序列克隆法从亚麻中克隆了雄性不育基因同源序列MS2-F cDNA(登陆号:EU363493).该cDNA全长1 91lbp,包含一个1 608 bp的ORF,编码535个氨基酸.推导的蛋白质序列中包含2个雄性不育保守区:NAD结合区域和雄性不育C-末端区域.该基因与油菜和拟南芥雄性不育基因的一致性分别为59.65%和59.16%,为花蕾特异表达基因,推测在亚麻花粉发育过程中与脂酰辅酶A还原酶有相似功能.MS2-F cDNA对应的gDNA大小为2 696 bp(登陆号:EU365361),含有8个内含子和9个外显子.     

9个辣椒雄性不育材料花蕾生理生化特性研究

以9个辣椒雄性不育材料及其相应的保持系为试材,对有关花器的植物学性状进行了系统观察和研究,测定了花蕾中过氧化物酶、过氧化氢酶、多酚氧化酶的活性和游离脯氨酸的含量。结果表明:供试9个雄性不育材料和保持系的主要花器性状有一定差异;雄性不育材料的过氧化物酶活性和多酚氧化酶活性均高于相应的保持系, 而过氧化氢酶活性低于保持系;保持系游离脯氨酸含量离于不育材料;所测的3种酶活性和脯氨酸含量在雄性不育材料与保持系间差异显著。     

白菜核雄性不育系可育和不育花药中Ca2+的分布

研究了白菜(Brassica campestris L. ssp.chinensis Makino)细胞核雄性不育系花药中Ca2 的分布特征.在可育花药发育过程中,减数分裂后花药壁细胞中钙颗粒明显增加.早期小孢子开始积累钙颗粒并特异性地附在小液泡膜上.小孢子分裂后,大液泡消失过程中又伴随着许多钙颗粒附在小液泡膜上,显示出Ca2 与花粉中液泡的形成和分解有关.在不育花药中,最早出现的钙颗粒异常分布是在小孢子母细胞的胼胝质壁中积累了较多的钙颗粒.然而,在小孢子细胞质中钙颗粒一直很少,也不形成大液泡,最后通过细胞质收缩的方式败育.这是首次发现Ca2 参与调控花药发育过程,其异常分布与花粉败育密切相关.     

A Comparison of Anther Tissue Development in Male Sterile Aloe vera and Male Fertile Aloe ciliaris

Cytological differences between the anther development of amale sterile and a male fertile Aloe species are used to explaininteractions between anther tissues. Some deviations in thelayers of the locule wall and the microspores of the male sterileanther are related to each other and their biological functionsare discussed. The cytological development of the male sterility,which can be observed shortly after meiosis, seems to be restrictedto the locular cavity. The tapetal development and breakdownare normal, apart from the size of some orbicules. However,the pollenkitt is not transported to the pollengrains, whichstrongly supports our theory that this process is mechanicallypollen-controlled. The development of the epidermal and endothecialcells is normal, except in a part of the anthers where thesecells do not expand, after which dehiscence is incomplete. Thelatter process is discussed in relation to the deviations insidethe locular cavity. Aloe vera (L.) Burm. fil., Aloe ciliaris Haw., Liliaceae, male sterility, tapetum, pollenkitt, endothecium, anther dehiscence     

棉花洞A雄性不育系花药发育的mRNA差别显示

应用cDNA-AFLP(Amplified fragment length polymorphism）技术,分析了棉花洞A雄性不育和可育材料在花药发育过程中花粉发育相关基因表达的差异。结果表明：在花药发育的相同时期,不育和可育花粉cDNA-AFLP的谱带在单核期的差异大于减数分裂期。在花粉发育过程中,花粉发育相关基因的表达具有时空特异性。共回收了64条差异cDNA片段,随机选择3个片段标记为探针,RNA斑点杂交分析表明,GHA27具有花器官组织特异性,仅在花器官中表达;GHA28、GHA47则具有花药组织特异性且仅在可育花药中表达。序列相关性比较表明：GHA27与植物ADP-ribosylation factor(ARF）基因有较高的相似性,而GHA28、GHA47与已知序列的相似性很低。     

Differential Mitochondrial Electron Transport through the Cyanide-Sensitive and Cyanide-Insensitive Pathways in Isonuclear Lines of Cytoplasmic Male Sterile, Male Fertile, and Restored Petunia

Three pairs of isonuclear lines of cytoplasmic male sterile (CMS) and fertile Petunia cells (Petunia hybrida [Hook] Vilm. and Petunia parodii L.S.M.) grown in suspension culture were examined for sensitivity to inhibitors of respiratory electron transport at time-points after transfer into fresh media. Cells from CMS lines differed from cells of fertile lines in their utilization of the cyanide-insensitive oxidase pathway. Under our culture regime, after approximately 3 days of culture cells from the CMS lines exhibited much lower cyanide-insensitive, salicylhydroxamic acid-sensitive respiration than cells from the fertile lines. This respiratory difference was shown to be specific to the mitochondrial alternative oxidase pathway by using other characteristic inhibitors of mitochondrial electron transport in experiments with isolated mitochondria. Immature anthers from CMS plants also showed lower alternative oxidase activity relative to anthers from male fertile plants, but no such difference was detected in leaf tissue, ovary or perianth tissue, or anthers collected just prior to anthesis. A cell line from a fertile plant carrying a nuclear fertility restorer gene and the CMS cytoplasm exhibited increased activity of the alternative pathway compared with the CMS lines.     

A Comparative Study of Anther and Pollen Development in Male Fertile and Male. Sterile Green Onion (Allium fistulosum L.)

Anther and pollen development in male-fertile and male-sterile green onions was studied. In the male-fertile line, both meiotic microspore mother ceils and tetrads have a callose wall. Mature pollen grains are 2-celled. The elongated generative cell with two bended ends displays a PAS positive cell wall. The tapetum has the character of both secretory and invasive types. From microspore stage onwards, many oil bodies or masses accumulate in the cytoplasm of the tapetal cells. The tapetum degenerates at middle 2-celled pollen stage. In male-sterile line, meiosis in microspore mother cells proceeds normally to form the tetrads. Pollen abortion occurs at microspore with vacuole stage. Two types of pollen abortion were observed. In type I, the protoplasts of the microspores contract and gradually disintegrate. At the same time the cytoplasm of microspores accumulates oil bodies which remain in the empty pollen. The tapetal cells behave normally up to the microspore stage and early stage of microspore abortion, but contain fewer oil bodies or masses than those in the male-fertilt line. At late stage of microspore abortion, three forms of the tapetal ceils can be observed: (1) the tapetal cells with degenerating protoplasts become flattened, (2) the tapetal cells enlarge but protoplasts retractor, (3) the cells break down and tile middle layer enlarges. In type Ⅱ, the cytoplasm degenerates earlier than the nucleus of the microspores and no protoplast is found in the anther locule. There are fibrous thickenings iii the endothecium of both types. It is difficult to verify whether the tapetum behavior and pollen abortion is the cause or the effect.     

苜蓿雄性不育植株与可育植株现蕾初期花蕾蛋白质组比较

采用双向凝胶电泳对现蕾初期苜蓿雄性不育植株(Ms-4)及其可育植株(MF)花蕾蛋白质进行了分离,获得了分辨率和重复性较好的双向电泳图谱。通过ImageMaster 2D软件对Ms-4和MF银染图谱分析发现,两者在等电点5~7、分子量20~60 kD范围内蛋白质斑点分布最多,可识别的总蛋白质点数均在6 000个左右,其中差异表达的蛋白质点数为98个;进一步通过质谱分析成功鉴定了22个差异蛋白点。利用Blast2GO程序对 22个蛋白点进行功能注释和代谢途径分析发现,核酮糖羧化酶小亚基、尿苷三磷酸-葡萄糖-1-磷酸尿苷酰基转移酶等蛋白在光合作用、碳水化合物代谢、多细胞生物有机体的发育等过程中起着重要的作用,同时参与了细胞质、细胞壁等组成,并具有绑定、催化、结合和水解等功能。研究结果初步推断,在苜蓿花药发育过程中,蛋白的缺失及表达量的变化可能会使与花粉发育有关的能量缺失,物质合成发生改变,导致雄性不育。