光周期敏感核不育水稻叶绿体的特异性蛋白质

用双向聚丙烯酰胺凝胶电泳技术,将光周期敏感核不育水稻“农垦58S”和其对照品种“农垦58”苗期及育性转换光周期敏感期的叶绿体蛋白质分离为大约90个蛋白质点。“农垦58S”的叶绿体内有一个45kD(pI_(6.7))和一个61kD(pI_(6.0))的特异性蛋白质点,而“农垦58”没有。“农垦58S”的另一个61kD(pI_(6.2))蛋白质点的含量明显高于“农垦58”。不同光周期(长日照和短日照)处理不影响光敏感期的这种叶绿体蛋白质的差异。     

Relationship Between Male Fertility and Endogenous Phytohormones in Photoperiod-Sensitive Genic Male-Sterile Rice

A spontaneous male sterile rice plant (Oryza sativa L. cv. Nongken 58S) "Photoperiod-sensitive Genic Male-sterile Rice has been found to be male sterile under long day cycles (LD) and fertile in short day cycles (SD). The period from secondary rachis-branch and spikelet primodia to pollen mother cell formation in the process of panical development was the photoperiod-sensitive stage for fertility alternation. The phenotype of this mutant was reported to be controlled by two pairs of recessive alleles. The research on relationship between the fertility alternation and phytohormone action in this mutant have been performed by Chinese scientists since 1985. In order to study the mechanism of fertility alternation in Nongken 58S, endogenous IAA, ABA, GA1 and GA4 in apical leaves and reproductive organs in different development stages under LD and SD conditions have been quantiatvely and qualitatively identified by GC-MS-SIM method. It was found that endogenous IAA in apical leaves at the stage of pistil and stamen primodia formation and in panicles at pollen mother cell stage of Nongken 58S with LD condition was deficient comparing with those in SD. Endogenous ABA level in panicles at pollen mother cell stage, in spikelets at uninucleate stage and in anthers at anthesis stage of Nongken 58S-LD were lower than those in SD. ABA levels in corresponding organs and developmental stages of wild type of rice, "Nongken 58" were always higher in LD treatment than those in SD. Endogenous IAA, GA1 and GA4 levels in anthers at anthesis stasge of "Nongken 58"-LD were increased obviously. Thus it appeared that "Nongken 58" possess stronger resistance to LD stress than Nongken 58S. It is concluded that IAA deficiency of reproductive organs at early developmental stage, ABA decrease implying poor resistance to LD stress and reduction of GAs in late developmental stages were the factors causing the anther sterility in Nongken 58S-LD.     

In Vitro Culture of Different Explants from Hubei Photoperiod Sensitive Genic Male-Sterile Rice

Young panicles, immature embryos, stem nodes, stem tips, leaf segments, root tips and anthers from Hubei Photoperiod Sensitive Genie Male-Sterile Rice (Oryza sativa subsp, japonica) “Nongken 58” (abbr. 58s) and fertile “Nongken 58” (abbr. 58f) were examined for callus induction, plant regeneration and direct plantlet formation on differentiation medium. 58s and 58f had equal ability in all explants cultures except anther cultures. The induction frequency of the anther callus and the regeneration frequency of the green plant in 58s were much lower than those in 58f, and such differences were not affected markedly by the change of fertility of 58s donor plants. Young panicle, immature embryo, stem node, stem tips showed direct plantlet formation when cultured on differentiation medium containing NAA and kinetin. Different explants produced various types of responses. Young panicles could produce callus and then regenerate plantlets. Evidences from histological observation showed that the plant regeneration in direct plaatlet formation of young panicles were mainly organogenetic, bowever, somatic embryogenesis was also possible.     

Analysis on Amino-Terminal Sequence and Purification of a 61 kD Protein from Photoperiod-Sensitive Genic Male-Sterile Rice

The specific protein P2,one of the three specific proteins(P1,P2 and P3)in chloroplasts from photoperiod-sensitive genlc malesterile rice previously reported was purlfied through preparative two dimensional gel electrophoresis and preparatwe isoelectric 10-CUSlng(1EF) and from which an uniform P2,checked with SDS-PAGE and IEF,was obtained．The molecular weight and isoelectric point was 61 kD and 5,8,respectively．Therefore P2 was referred as P61．A search in databases revealed that the aminoterminal sequence Of P61 was identical to that of perb unit of chloroplast ATP ase from barley and rice     

Energy and Activated Oxygen Metabolisms in Anthers of Hubei Photoperiod-Sensitive Genic Male-Sterile Rice

The energy and activated oxygen metabolisms in male-sterile and fertile anthers of Hubei photoperiod-sensitive genic male-sterile rice (HPGMR)have been comparatively studied. Among the male-sterile anthers, lower total activities of cytochrome oxidase(COD), ATPase, peroxidase (POD), catalase (CAT) and superoxide dismutase(SOD) in six anthers of a spikelet were seen, which were equivalent to about 2–64%, 20–76%, 26–60%, 6–77%, 29–55%, respectively, of those of their fertile counterparts during different development stages of pollens. The sterile anthers lacked 1–5, 1, 1–2 isozyme bands of COD, POD and SOD, respectively, and also displayed lower content of ATP, higher contents of H202 and malondiald ehyde (MDA) and higher efficiency of O2- which were equivalent to about 14–77%, 152–424%, 153–238%, 230–340%, respectively, of those of their corresponding fertile ones. It is concluded that the physiological features of male-sterile anthers, viz. lower efficiency of oxidative phosphorylation, higher efficiency of O2 and H202 generation, weaker activity of scavenger system of activated oxygen and higher level of lipid peroxidation, are indicative of some relationship between abnormal energy, activated oxygen metabolisms in male-sterile anthers and male-sterility in HPGMR.     

RAPD在水稻温敏核不育研究的应用

ＲＡＰＤ是新发展起来的一种分子标记技术,近来得到广泛应用,我们对这种技术的实验程序进行了研究,摸索出了ＲＡＰＤ反应的适宜条件,并在不影响扩增结果的前提下,缩短反应时间,提高了仪器的利用率,有利于大量样品的分析,进而用这种技术对温敏核核不育水稻安农Ｓ－１及其原始株进行分析,在２００个引物中发现了１个引物在２种材料中扩增带型有差异,并初步认为此差异与不育相关。     

Effect of Photoperiod Treatments on Phytochrome a Levels and Its mRNA Abundance in Photoperiod-Sensitive Genic Male-Sterile Rice Mutant and the Wild Type

The effect of photoperiod treatments on phytochrome A (Phy A) level and its mRNA abundance in the leaves of a photoperiod-sensitive genic male-sterile mutant (Nongken 58S) and its wild type ("Nongken 58') of Oryza sativa L. was investigated. The top two leaves of each rice shoot were harvested at the end of the last dark phase of 10 cycles during photoperiod-sensitive stage for fertility alteration of the mutant. Phy A was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal and mouse monoclonal antibodies. Compared with longday (LD) treatment,short day (SD) resulted in 38.5% increase of relative Phy A content in the mutant, only 18.5% increase in the wild type. In an extended darkness (25 h), the accumulation of Phy A also appeared to be more rapid in the mutant seedlings than in its wild type. RNA dot blot analyses with RPA3 (a cDNA clone of rice Phy A) as a probe showed:the abundances of Phy A mRNA in top leaves of Nongken 58S and "Nongken 58" were obviously higher in SD than those in ID at the end of dark phase of 5 d and 10 d photoperiod treatments. Moreover, under SD Phy A mRNA contents in Nongken 58S were more than those in "Nongken 58" during the whole photoperi- od-sensitive stage for fertility alteration. In addition, after 10 cycles of end-of-day far-red irradiations (EOD FR), the heading and flowering date of the mutant was delayed for 2 d. However, EOD FR had little or no effect on male fertility of the mutant.     

Studies on Photoperiod-sensitive Genic Malesterile Rice in Terms of Developmental Biology

Investigations on the mechanism of fertility alternation and its regulation in photoperiodsensitive genic male-sterile rice (PGMR) have received considerable attention due to the important significance in both basic research of plant developmental biology and PGMR potential value in the development of two line hybrid rice seed programs. The present review described the major achivements of study on PGMR in terms of developmental biology: phytochrome was found to be the photoreceptor involved in photoperiod-modulated fertility alternation in PGMR; chloroplasts played a significant role in the photoperiodic signal transduction; gibberellins and auxin might be the chemical signals for fertility regulation; identifying of special proteins promoted the exploration of specially expressed genes related to fertility regulation in PGMR. The author hypothesized that four main steps of photoperiodic signal transduction were involved in the realization of male sterility-multistage magni-fied injury effects to normal function of PGMR induced by long-day stress. There was defect in the anther development of PGMR, and the resistance of anthers to environmental stress was weakened. The multifactor coaction model for photoperiod regulation in fertility alternation in PGMR was proposed and discussed.     

Effects of Al3+ on Ca2+-ATPase Activity on Chloroplasts of Rice and Relation to Calmodulin

The relationship between aluminium phytotoxicity and calmodulin has been studied with. calcium-dependent ATPase in chloroplasts of rice. This enzyme could be activated by extrinsic calmodulin. It showed that the activity of Ca2+-ATPase in chloroplasts was regulated by calmodulin. The activation of calmodulin to the enzyme might be inhibited by calmodulin antagonists, TFP and CPZ. The effects of A13+ on the activation of calmodulin was similar to that of the calmodulin antagonists. Calcium could reduce the inhibition of aluminiutn. It seems that there is a model of toxic responses in plants to aluminium: Al3+→calmodulin→target enzy mes→metabolism.     

Characterization of the Membrane- Bound Gibberellin Binding Proteins from Young Shoots of Rice

Gibberellin-binding proteins were found on the membrane of young rice shoot. The dissociation constant (Kd) for GAs was approximately 6.5 × 10-8 mol/L, and the total concentration of the sites was 0. 3 pmol ·mg-1 protein. The binding activity of gibberellin-binding proteins was significantly affected by temperature and phi which was 140% higher at 0 ℃ than that at 25 ℃, and the optimal pH value was 5. Gibberellin-binding activity increased with the incubation time, reaching the maximum at 1 h. and then decreased gradually. Both IAA and ABA were able to compete with GA3 for gibberellin-binding proteins.     

光周期对光敏胞质不育小麦花药发育过程中Ca^2＋分布的影响

运用焦锑酸钾沉淀法,研究了不同光照条件下光敏胞质不育小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）花药发育过程中Ｃａ＾２＋的分布。短日照条件下,小孢子形成和花粉发育过程中胞内Ｃａ＾２＋在数量、分布上有变化;小孢子表面逐渐积累Ｃａ＾２＋,至成熟花粉表面覆盖一层Ｃａ＾２＋,胞质Ｃａ＾２＋较少;药隔和药壁组织通过抽外体或共质体途径运输Ｃａ＾２＋供给花粉的发育;长日照条件下,花粉败育发生在不同时期,早期     

水稻稻瘟菌抗性相关蛋白的双向电泳分析

建立抗感水稻品种受稻瘟菌侵染和未侵染蛋白质的双向凝胶电泳图谱,分析其差异表达的蛋白,寻找稻瘟病的抗性相关蛋白,以阐明稻瘟病的发病机制。采用TCA/丙酮沉淀法提取四种愈伤组织材料的总蛋白并采用固相pH梯度( immobilized pH gradient, IPG)双向凝胶电泳( two-dimensional gel electrophshiya, oresis, 2-DE)分离四种材料总蛋白质, 凝胶经银染显色后,用PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质。成功获得抗感水稻品种受稻瘟菌侵染和未侵染蛋白质的双向凝胶电泳图谱。获得未侵染内香优2号平均蛋白点数为447个,汕优63平均蛋白点数为440个;侵染后抗性品种内香优2号平均蛋白数为523个,感性品种汕优63平均蛋白质点数为326个。内香优2号未经侵染和侵染后图谱匹配率达 89%和 87%,汕优63未经侵染和侵染后图谱匹配率达86％和85％。内香优2号的差异表达蛋白点数为76个,汕优63的差异表达蛋白点数为114个。两者间存在一些差异表达的蛋白质,为阐明稻瘟病的发病机制打下了坚实的基础。     

乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化

应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27．1kD、p,7．3),TE2(17．5kD、pI8．2)2个特异蛋白,45d存在TE3(11．4kD、pI7．6),TE4(13．2kD、pI9．9)2个特异蛋白,52d存在TE5(22．6kD、pI7．2),TE6(18．6kD、pI8．3),TE,(23．5kD、pI3．6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失．对胚胎的分化发育具有重要作用。     

温敏雄性不育水稻培矮64S花药发育过程中钙的变化

采用焦锑酸钾沉淀法研究了温敏雄性不育水稻(Oryza sativa L.)培矮64S在高温引起雄性不育与正常可育花药发育过种中Ca2+的分布变化.结果表明,当培矮64S生长在较高温度条件下引起雄性不育,与可育花药相比,不育花粉母细胞中有较多的液泡、较多的Ca2+沉积和较少的线粒体,并且有较多的Ca2+沉积在不育花药的中间层、表皮层和绒毡层中.到四分体与单细胞花粉时期,不育花药的木质部细胞的次生加厚壁上有较多的Ca2+沉淀,连接组织中的Ca2+沉淀也大大增加,所有不育花粉外壁较厚而发育都不正常.在单核细胞早期,不育花粉的四分体细胞中有较明显的大液泡出现.不育花药中的Ca2+在花药发育的各时期均比可育花药要多.这些结果说明在高温生长条件下,花粉母细胞发育的异常、花药中Ca2+沉积的增加、绒毡层与花粉外壁发育的异常可能与培矮64S花粉败育相关.     

Abnormality in Male Organ Development and Fertility of Photoperiod sensitive Genic Male Sterile Rice Plants Under Short Day Condition

Ten photoperiod-sensitive genic male sterile rice (PGMSR) lines were studied to observe the development of male organs of their short day (SD) plants. The results were shown as follows: 1 ) The self seed-setting rate and the percentage of fertile pollen of these PGMSR lines were much lower than those of the controls. 2) All the male sterile line plants had variant barriers in anther dehiscing and pollen scattering. In addition, some of their stigmas were devoid of pollen, and some self pollen grains on their stignas germinated weakly. 3) Female fertility of those plants was believed to be normal through observation in their embryo sac development and cross seed-setting rate. The authors deduced that the male sterile gene expression becomes the major factor for low rate of self seed-setting of PGMSR plants under SD condition.     

水稻叶绿体基因文库的构建和精细限制图谱的制作

水稻幼叶在加有高浓度抗坏血酸的缓冲液中匀浆,以获得完整的叶绿体,从中分离到ctDNA得率高达100μg/100g叶,纯度足以用于限制性核酸内切酶分析。ctDNA经Mbo I部分酶解得到的片段克隆到载体pcos 2 EMBL的Bam HI位点,重组DNA经体外包装后感染宿主菌,筛选表型Tc~5Km~R的重组子,通过计数克隆有效率达5×10~4重组菌落/1微克插入DNA。用λ-末端酶对重组环状双链DNA在cos位点切成线性分子,产生两个(ON-L及ON-R)可供标记和杂交的末端,线性Cosmid DNA经限制酶部分消化,凝胶电泳分离,干燥凝胶放射自显影,得到了6种限制性核酸内切酶的限制图谱。水稻ctDNA全长为129.5kb,在ctDNA上Pvu Ⅱ、Sal Ⅰ、Pst Ⅰ、Hind Ⅲ、Eco RI及Bam HI的切点分别为11、12、17、37、67和44个,1R A和B为21.7kb,LSC为73.7kb,SSC为12.4kb。     

水稻叶绿体16S启动子克隆改造、载体构建及转化研究

利用PCR方法从水稻叶绿体基因组DNA中分离16S启动子,并在其下游加入rbcL基因SD序列,以增强该启动子的翻译能力;序列分析表明,除加入的SD序列外,扩增片段与水稻(Oryza sativa)叶绿体基因组DNA序列16S启动子相应区域同源性为100％。将16S启动子与bar基因和gfp基因的融合基因连接,以psbA基因的3′序列为终止子,并以烟草叶绿体trnH—psbA和trnK为同源片段构建了烟草叶绿体表达载体pRl6S。用基因枪转化烟草,转化植株经Southern、Northern检测及后代遗传学分析,发现：16S启动子具有启动活性,融合基因已在烟草叶绿体中稳定整合并遵循母系遗传规律。     

利用初级三体定位水稻光敏核不育基因

自 1 973年 Ramanunjam首次获得三体 ( 2 n=2 x 1 =2 5)植株以来 ,已报道的粳型初级三体有日本晴 NT和籼型初级三体 IR36、Sona、广陆矮 4号 [1 ]及籼型初级三体 30 37[2 ]等。获得细胞学证据并用于定位研究的仅有日本晴三体和 IR36三体 [3,4]。目前 ,利用形态标记和分子标记定位水稻光敏核不育基因的报道较多 ,且涉及到第 5、7和第 1 2三条染色体 [5,6] 。我们拟以粳型光敏核不育系为父本 ,台中 65初级三体为母本配组 ,定位光敏核不育基因 ,对粳型光敏核不育基因定位结果进行验证 ,以期为光敏核不育的利用与研究提供理论依据。1 　材料…     

Two Types of FtsZ Proteins in Mitochondria and Red-Lineage Chloroplasts: The Duplication of FtsZ Is Implicated in Endosymbiosis

The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.