烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚.要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料.以前的研究表明,烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显.这种异型性的精细胞可能与倾向受精有关.烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞.用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞.用微量酶液可使两个精细胞分开.然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体.这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础.本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件.     

烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚。要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料。以前的研究表明, 烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显。这种异型性的精细胞可能与倾向受精有关。烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞。用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞。用微量酶液可使两个精细胞分开。然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体。这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础。本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件。     

异胡豆苷合成酶在烟草亚细胞区室的表达(英)

异胡豆苷合成酶 (strictosidinesynthase,STR)是吲哚生物碱生物合成的一种关键酶 ,将色胺 (tryptamine)和裂环马钱子 (secologanin)耦合成为吲哚生物碱的前体化合物异胡豆苷。将异胡豆苷合成酶标定在烟草植物不同的亚细胞区室———叶绿体、液泡和内质网中表达 ,通过蛋白免疫印迹分析和STR酶活性的测定 ,表明STR在叶绿体、液泡和内质网中有效表达。STR体外酶活性分析采用间接荧光法检测色胺在反应体系的消耗。STR的酶活性分析表明了STR在烟草中不同的亚细胞区室得以活性表达。分离纯化转基因烟草的叶绿体 ,通过对其分离的不同部分的蛋白免疫印迹分析 ,确定了将STR正确标定在烟草的叶绿体中表达。     

异胡豆苷合成酶在烟草亚细胞区室的表达

异胡豆苷合成酶(strictosidine synthase,STR)是吲哚生物碱生物合成的一种关键酶,将色胺(tryptamine)和裂环马钱子(secologanin)耦合成为吲哚生物碱的前体化合物异胡豆苷.将异胡豆苷合成酶标定在烟草植物不同的亚细胞区室--叶绿体、液泡和内质网中表达,通过蛋白免疫印迹分析和STR酶活性的测定,表明STR在叶绿体、液泡和内质网中有效表达.STR体外酶活性分析采用间接荧光法检测色胺在反应体系的消耗.STR的酶活性分析表明了STR在烟草中不同的亚细胞区室得以活性表达.分离纯化转基因烟草的叶绿体,通过对其分离的不同部分的蛋白免疫印迹分析,确定了将STR正确标定在烟草的叶绿体中表达.     

羟自由基诱导烟草细胞凋亡

用适当浓度组合的硫酸亚铁和过氧化氢反应以产生羟自由基来处理烟草悬浮细胞,首次观察到了细胞中染色质凝缩、边缘化、细胞核解体等典型的细胞凋亡的形态学特征;在ＤＮＡ 琼脂糖凝胶电泳图谱上观察到因核ＤＮＡ 有规则地在核小体间被降解而产生的阶梯状条带;末端脱氧核糖核酸转移酶介导的３’ＯＨ 末端标记法（ｔｅｒｍｉｎａｌ ｄｅｏｘｙｎｕｃｌｅｏｔｉｄｙｌ ｔｒａｎｓｆｅｒａｓｅ （ＴｄＴ）ｍｅｄｉａｔｅｄ ｄＵＴＰ ｎｉｃｋｅｎｄｌａｂｅｌｉｎｇ,ＴＵＮＥＬ） 检测结果为阳性。这些实验表明,羟自由基确能诱导烟草细胞凋亡。     

烟草初生胚乳细胞在微室培养中的发育

烟草初生胚乳细胞在微室培养中的发育 李师弢 房克凤 杨弘远*     

重组色氨酸脱羧酶在烟草不同亚细胞区室的表达

将萜烯类吲哚生物碱代谢关键酶--色氨酸脱羧酶（TDC）的编码基因转到烟草（Nicotiana tabacum L.)植物体内，标定在不同的亚细胞区室表达。通过蛋白免疫印迹法和色胺在植物体内的累积量测定分析，对转基因植物进行筛选。结果表明，TDC在叶绿体和胞液中高效表达，TDC在叶绿体中的表达水平最高，高于在胞液中的表达，在内质网和液泡中表达水平很低，用蛋白免疫印迹法未检出。     

重组色氨酸脱羧酶在烟草不同亚细胞区室的表达

将萜烯类吲哚生物碱代谢关键酶———色氨酸脱羧酶 (TDC)的编码基因转到烟草 (NicotianatabacumL .)植物体内 ,标定在不同的亚细胞区室表达。通过蛋白免疫印迹法和色胺在植物体内的累积量测定分析 ,对转基因植物进行筛选。结果表明 ,TDC在叶绿体和胞液中高效表达 ,TDC在叶绿体中的表达水平最高 ,高于在胞液中的表达 ,在内质网和液泡中表达水平很低 ,用蛋白免疫印迹法未检出。     

Surface Topography and Cytomatrix Elements in Protoplasts from Plant Cells

We studied the surface topography of protoplasts from callus of Daucus sativus(Hofm.) Roehl. and from mesophyll cells of Nicotiana tabacumL. We also followed the distribution of actin elements of the cytomatrix in the subcortical cytoplasm layer. Protoplasts were prepared for scanning electron microscopy by a modified method without drying in the critical point apparatus. After postfixation with OsO₄, carrot and tobacco protoplasts had a similar topography of the surface: it was rough and had few pores. When carrot protoplasts were not postfixed with OsO₄, their surface looked different: it was folded and had 1.5-m pores, which sometimes were bordered with globules 0.3 m in diameter, or it consisted of conical cells varying in depth and size of their bases. We believe that, when protoplasts were not fixed with OsO₄, they lost lipid-containing structures from their surface, and what remained was the protein carcass of the plasmalemma and the underlying layer of the cytoplasm. The inner surface of opened carrot protoplasts had elaborate topography, apparently produced by the elements of the cytomatrix, that is, a relatively thick layer of the cortical cytoplasm, where, using phalloidin–colloidal gold and transmission electron microscopy, we could observe a dense network of actin filaments.     

烟草株高变异体的茎尖中内源激素含量变化及其对外源激素的响应

烟草矮秆基因型“农大202’的茎生长过程中茎尖中内源赤霉素（GA3）、生长素（IAA）含量始终低于中、高秆基因型,其内源脱落酸（ABA）和玉米素（ZR）含量则相对较高。喷施外源激素GA,和IAA的结果表明,GA,可调节矮秆基因型茎尖中各内源激素的含量,与正常株高基因型‘K326’的各激素含量相接近,从而促进茎生长,而IAA的作用较小。     

Resistance Mechanism of Cultured Plant Cells to Tobacco Mosaic Virus

In somaclonal tissues obtained from systemically TMV-infected tobacco plants, a relation between changes of TMV amounts and the callus growth was examined. The culture medium was suitable for maintaining a constant concentration of TMV as well as active callus growth. By using the shake-culture method, somaclonal tissues were separated into two classes on the basis of callus sizes. In large callus tissues, TMV amounts were constant during subculturing but the tissues did not either grow or release the newly divided cells after the last subculture. On the other hand, smaller callus tissues grew markedly and the TMV amounts were conspicuously lowered. After shake-subculture of smaller tissues, they were successfully regenerated to plantlets. None of the plantlets expressed any mosaic symptoms, while plantlets from the original somaclones showed severe mosaic symptoms of TMV in leaflets. Thus, the present report describes the successful production of virus-free plantlets from infected somaclonal callus cultures.     

烟草细胞乙醇分解代谢能力的离体调控(英文)

报告了一些重要因子对培养在含有乙醇的培养基上的烟草细胞的乙醇分解代谢能力（ＥＣＡ）的调控效果。在供试的５种生长素和２种细胞分裂素中,ＩＢＡ４ｍｇ／Ｌ和Ｋｔ０．０５ｍｇ／Ｌ最有利于促进ＥＣＡ,而ＮＡＡ２ｍｇ／Ｌ和Ｋｔ０．０２５ｍｇ／Ｌ则最有利于支持处在乙醇胁迫下的细胞的增殖。实验中还发现提高维生素浓度有利于细胞的ＥＣＡ。通过将细胞从低乙醇浓度的培养基转移到较高乙醇浓度的培养基中,能够诱导细胞耐受更高浓度的乙醇并增强细胞的ＥＣＡ。研究结果可以应用于制备适合分离乙醇分解代谢途径中的酶的烟草细胞材料。     

Stomatal Morphology during Acclimatization of Tobacco Plantlets to ex vitro Conditions

Image analysis was used in studying stomatal morphology during acclimatization of tobacco plantlets to ex vitro conditions, 45 d after transfer leaf area was 15 times, and total number of stomata per leaf four times increased. During acclimatization stomatal density was decreased considerably on both leaf sides, and was compensated by an increase in stomatal sizes, e.g., in stomatal length and in stomatal area (both guard cells and pore). Elongation of stomata was increased indicating that the originally circular stomata of in vitro plantlets were changed into elliptical ones in ex vitro acclimatized plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

低温胁迫下褪黑激素对烟草悬浮细胞精氨酸脱羧酶活性的影响

研究了褪黑激素对烟草(Nicotiana tabacum)悬浮细胞在低温胁迫下精氨酸脱羧酶活性及细胞生存率的影响.发现褪黑激素可以明显提高低温胁迫下烟草悬浮细胞精氨酸脱羧酶的活性,并明显提高细胞的生存率.表明褪黑激素可能在低温条件下通过调节植物细胞内多胺的合成而提高抵御冷害的能力.     

外源生长素对烟草(Nicotiana tabacum L.)小孢子早期胚胎发生的影响

烟草（Nicotlana tabacum L．）小孢子胚胎发生系统,不仅可以提供大量可供处理的小孢子胚,还由于小孢子胚胎发生的不同步性,可同时提供从2-3细胞原胚到分化胚一系列胚胎以供研究。利用这一便利系统,探讨了外源生长素处理对小孢子胚胎发育的影响。使用3种浓度的IAA：1、3、10μmol／L,分别对不同发育时期烟草小孢子胚进行了处理,结果发现,对不同发育时期的小孢子胚,生长素处理的效果明显不同。外源生长素对胚胎发生有促进作用,表现为2-3细胞比例与非处理组相比升高,而当小孢子发育到小球形胚后,加入外源生长素对小孢子胚的进一步发育却表现出明显抑制作用。这说明在小孢子胚胎发育过程中早期和晚期发育对生长素的需求是不同的,且对生长素的敏感程度亦不同。反映了生长素调控机制在两个不同发育时期的差异。     

一种适于植物幼胚mRNA整体原位杂交的方法

A mRNA whole-mount in situ hybridization method is reported here for quick, direct analysis of the spatial and temporal mRNA expression patterns in plant young embryos. A cDNA clone THE3 (tobacco heart embryo 3) was isolated by differential screening from tobacco (Nicotiana tabacum L.) heart embryo cDNA library as compared with the globular embryo cDNA library. The distribution of THE3 mRNA in tobacco heart embryos and globular embryos was investigated by a whole-mount in situ hybridization technique, showing that THE 3 is preferentially expressed in heart embryos.     

Protein Kinase Activity of in vitro Cultured Plant Cells in Relation to Growth and Starch Metabolism

Taking tetcyclacis, a norbornenodiazentine derivative, as an example, the influence of a growth retardant on the shoot growth of sunflower, soybean, and maize seedlings grown and treated in hydroculture was investigated. In detail, the reduction in the length of various shoot sections {epicotyl, 1st internode, leaf blade) caused by the retardant was studied. At low concentrations of the retardant (\lt10^-6 M) the shortening effects are substantially attributable to an influence on cell elongation, whereas cell division is inhibited as the concentration increases (τ10^-6 M). A comparison of the effects of tetcyclacis in cell suspension cultures of appropriate plant species showed that also in this system concentrations τ 10^-6 M inhibited cell division growth, i. e. there is comparability of plant/ cell culture regarding the retardant effect on cell division. In contrast to the intact plants, however, cell elongation appears to be of only subordinate importance for the growth of cell cultures, as it has been shown using parsley cell suspension cultures.It is discussed to what extent influencing the gibberellin or sterol biosynthesis by means of tetcyclacis provides an explanation for the concentration-dependent effect on the cell division and cell elongation processes.     

The Relationship between Glycine Oxidation and Nitrate Reduction in Tobacco Leaves

The relationship between glycine oxidation and nitrate reduction was studied using tobacco (Nicotiana tabacum L.) leaf disks and reconstituted system of isolated mitochondria and NR (Nitrate reductase). It was found that glycine, either vacuum-infiltrated in to leaf disks or added to the reconstituted system, could increase the rate of nitrate reduction. The stimulating effect of glycine on nitrate reduction was greatly influenced by preillumination treatment of tobacco leaves, and also by the activity of respiratory chain. The rate of glycinedependent O2 consumption by mitochondria was lowered when KNO3 and NR were added to the system. It was also found that the activity of glycine decarboxylase increased with increase in nitrate concentrations in the sandculture medium. It was concluded that oxidative decarboxylation of glycine in mitochondria of leaf cells of C3 plants could provide NADH for nitrate reduction in cytoplasm in the light, and nitrate reduction and glycine oxidation were influenced by each other.     

Induction of Expression Instability of the nptII Gene in Transgenic Tobacco Plants

A comparative analysis of the neomycin phosphotransferase (nptII) gene expression was performed in two groups of transformed tobacco plants, one of which included plants with direct and inverted tandem uidA gene repeats in the T-DNA insertion. This insertion of inverted repeats was shown to reduce the level of stable nptII gene expression to 20%, as compared with 65% in the control transformants. The level of unstable expression of this gene substantially increased (up to 71.4% vs. 5.5% in the control group) when homologous sequences were brought together with direct tandem repeats in the genome of hybrid plants.