Flow Cytometric Evidence for Hydroxyl Radical-induced Apoptosis in Tobacco Protoplasts

Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.

Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

In the present study, we focused on whether Intracellular free Ca^2＋ （[Ca^2＋],） regulates the formation of mltochondrlal permeability transition pore （MPTP） In H2O2-induced apoptosis In tobacco protoplasts. It was shown that the decrease In mltochondrlal membrane potential （△ψm） preceded the appearance of H2O2-Induced apoptosls; pretreatment with the specific MPTP Inhibitor cyclosporine A, which also Inhibits Ca^2＋ cycling by the mitochondria, effectively retarded apoptosls and the decrease In △ψm. Apoptosls and decreased △ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca^2＋ channel blocker lanthanum chloride （LaCl3） attentuated these responses. Chelation of extracellular Ca^2＋ with EGTA almost totally Inhibited apoptosls and the decrease In △ψmInduced by H2O2. The time-course of changes In [Ca^2＋]l In apoptosls was detected using the Ca^2＋ probe Fiuo-3 AM. These studies showed that [Ca^2＋]1 was Increased at the very early stage of H2O2-Induced apoptosls. The EGTA evidently Inhibited the Increase In [Ca^2＋]1 Induced by H=O=, whereas It was only partially Inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca^2＋ concentrations In tobacco protoplasts, which mainly results from the entry of extracellular Ca^2＋, to regulate mltochondrlal permeability transition. The signaling pathway of [Ca^2＋]1-medlated mltochondrlal permeability transition was associated with H2O2-Induced apoptosis In tobacco protoplaete.

Electrofusion of tobacco protoplasts in space

Electrofusion of evacuolated (N. Tabacum L. cvs. Gexin no1) and vacuolated (N. Rustic) tobacco mesophyll protoplasts was performed on the Chinese spacecraft (Shenzhou No. 4, from 30th Dec. 2002 to 6th Jan. 2003). The results showed that the frequency of bi-nucleated and mul-tinucleated protoplasts was significantly increased under microgravity. Compared with the control samples incubated on the ground, the viability of protoplasts incubated in space was much higher. In addition, the influence of altered gravity on carbohydrates was also observed. These results confirmed the effect of microgravitation on electrofusion of plant cell protoplasts.

Break of symmetry in regenerating tobacco protoplasts is independent of nuclear positioning

Nuclear migration and positioning are crucial for the morphogenesis of plant cells. We addressed the potential role of nuclear positioning for polarity induction using an experimental system based on regenerating protoplasts, where the induction of a cell axis de novo can be followed by quantification of specific regeneration stages. Using overexpression of fluorescently tagged extranuclear （perinu- clear actin basket, kinesins with a calponin homology domain （KCH）） as well as intranuclear （histone H2B） factors of nuclear positioning and time-lapse series of the early stages of regeneration, we found that nuclear position is no prerequi- site for polarity formation. However, polarity formation and nuclear migration were both modulated in the transgenic lines, indicating that both phenomena depend on factors affecting cytoskeletal tensegrity and chromatin structure. We integrated these findings into a model where retrograde signals are required for polarity induction. These signals travel via the cytoskeleton from the nucleus toward targets at the plasma membrane.

Laser-induced tobacco protoplast fusion

Laser tweezers can manipulate small particles, such as cells and organelles. When coupling them with laser microbeam selective fusion of two tobacco protoplasts containing some chloroplast was achieved. Physical and biological variables that affect laser trapping and laser-induced fusion were also discussed. The results show that the effect of chloroplast content and distribution on the yield of cell fusion is remarkable.

Plasmalemma hyperpolarity and culture of sense and antisense abp transgenic tobacco protoplasts

The relationship among transfer and expression of auxin binding protein gene ( abp ), auxin (NAA) induced plasmalemma hyperpolarity and sensibility to auxin during protoplast culture was studied by measuring transmembrane potential difference (Em) and culturing the protoplasts of sense and antisense abp transgenic tobacco. The concentration of NAA inducing the highest degree of hyperpolarity of sense abp transgenic tobacco protoplasts was lower than the control, and in protoplast culture, their sensibility to auxin increased. The concentration of antisense abp transgenic tobacco protoplasts was higher than the control, and in protoplast culture, their sensibility to auxin decreased. These results demonstrated that ABP synthesized in endoplasmic reticulum needed to transport to cell membrane and functioned there.

烟草原生质体的分离纯化

研究从烟草叶片分离原生质体的若干影响因素.通过酶解法降解细胞壁释放出原生质体,利用离心和蔗糖漂浮法从粗提取液中纯化出原生质体,并通过血球计数板计数来比较制备的效果.结果表明:酶的种类、质膜稳定剂、渗透压稳定剂、pH及酶解时间是影响原生质体制备的重要因素.纤维素酶含量2%,果胶酶含量1%,2-N-吗啉乙烷磺酸(MES)浓度50mmol/L,Ca2+与甘露醇作为渗透压稳定剂,pH6左右,温度28℃,酶解时间4h时原生质体的分离效果最佳.

用同步辐射X-射线荧光分析法研究感病烟草单细胞内微量元素变化

用同步辐射 X-射线荧光分析 (SRXFA)技术分析植物单细胞的微量元素 ,研究病毒侵染植物后植物单原生质体内微量元素的相对变化。用果胶酶和纤维素酶分离烟草叶肉细胞原生质体 ,经 3%戊二醛固定 30min后 ,置于杜乐膜上 ,干燥后用同步辐射 X-射线荧光法分析。 X-射线流强大于 80 m A时 ,停留取谱时间2 0 min,可从烟草单个原生质体中检测到 Cl,Ca,Mn,Fe,Cu,Zn,Cr,Co,Ni,As,Ti和 Ge。流强低于 80 m A时 ,只可检出 Cl,Ca,Fe,Cu,Ti和 Ge。结果表明烟草被烟草花叶病毒感染后 ,Ca,Fe离子的强度下降 ,Cl,Cu,Ge离子变化不显著。

烟草原生质体活力检测和细胞核染色方法的研究

本试验以烟草叶片和烟草悬浮系制备的原生质体为材料,采用不同染料对原生质体活力和细胞核进行染色效果比较.结果表明,原生质体活力观察宜采用荧光素双醋酸酯(FDA)染色法,染色效果清晰易于观察,四嘧唑蓝(MTT)法则可定量反映原生质体活力;相对于其他细胞核染料,DAPI对不同来源的原生质体细胞核染色效果均较好,能够清晰地对原生质体进行观察和计数.本研究为今后原生质体的检测提供有用的参考.

烟草原生质体分离纯化条件的优化

为提高烟草原生质体的产量和活力,对影响烟草原生质体分离纯化的酶解液组合、酶解时间、酶解渗透压、离心转速、基因型等因素进行了优化。结果表明:以1%纤维素酶+0.5%果胶酶+0.6mol/L甘露醇+0.1%MES的酶解液组合经6h酶解,700r/min离心5min得到的原生质体产量和活力相对较高;生理状态相近的不同烟草材料在产生原生质体的能力上存在一定差异。

烟草原生质体制备体系的优化及PVY^N导入研究

植物原生质体是除去细胞壁后仍具有活力的裸露单细胞,其在植物RNA病毒的复制机理研究方面具有广泛的应用。为了获得高产、高活力的烟草原生质体,以烟草(Nicotiana tabacum Linn.)品种K326为材料,对原生质体制备体系中的不同细胞壁酶解液成分、酶解时间、酶解渗透压稳定剂含量、离心时间以及离心转速等因素进行优化。结果表明:在含1%纤维素酶,0.5%离析酶,0.6mol·L-1甘露醇的酶解液中,26℃慢速震荡(40r·min-1)酶解4h,700r·min-1离心3min,得到产量及活力较高的原生质体,可直接用于植物病毒的接种试验;利用聚乙二醇(PEG)融合法向获得的高活力原生质体中接种马铃薯Y病毒N株系(Potato virus Y necrosis strain,PVYN),经培养后提取总RNA,通过PVYN特异性引物经RT-PCR扩增后检测条带明显,表明PVYN成功导入烟草原生质体并自我复制。

空间电融合的烟草原生质体再生植株分析

报告了在"神舟四号"飞船中通过电融合获得的烟草融合细胞,经地面培养获得再生植株。空间飞行样品再生愈伤组织的频率为地面对照的3倍多,而植株再生频率却比地面对照约低20%,约有32%的再生植株可能具有杂种性状。选取叶形状变化最为明显的H23号植株与黄花品种进行回交,回交一代的叶比原始材料宽,花的颜色与亲本黄花品种相同,表明空间微重力环境中融合的烟草原生质体能够再生植株,获得有繁殖能力的杂种。

烟草原生质体在植物病毒研究中的应用

烟草原生质体是植物病毒研究中应用最早、最广泛的原生质材料,它对植物病毒的研究起了有很大的推动作用。笔者介绍了它在植物病毒研究中的一般应用程序,综述了它在植物病毒的侵染、复制、病毒与寄主互作、转基因抗病毒、病毒抑制物等各方面研究中的应用。

玉米Ac双因子转座标签系统的构建及其转化烟草后代的遗传分析

用使质粒pKU3所携带的玉米Ac因子的5'末端和中间编码转座酶的基因片段分别发生缺失的方法构建成质粒pKU3（△B）和pKU3（△H）。质粒所携带缺失的Ac因子单独存在时都无自主转座能力，但当Ac（△H）和Ac（△B）共存于一个细胞时，由于Ac（△B）产生转座酶的互补作用促使Ac（△H）恢复转座能力，而当Ac（△B）和Ac（△H）因子被分离后即获得Ac（△H）因子的稳定插入突变株，它可克服因突变不稳定而给用转座因子标签法分离基因所造成的困难。将双因子系统导入烟草原生质体并获得再生植株，从而选得卡那霉素抗性植株，显示Ac（△H）因子已经从原质粒上的NPTⅡ前导顺序中切离。Sortharnblot和PCR分析表明：Ac（△H）和Ac（△B）因子已经整合在转化烟草的基因组并能遗传至F1和F2代植株中；有些植株后代中已检测不到Ac（△B）因子的存在，说明它们的Ac（△B）因子已与Ac（△H）因子相分离；各转化植株中Ac（△H）因子在基因组中的插入拷贝数从一个到几个不等；初步显示Ac（△H）因子多数插入或转座在基因组的结构基因中。

聚乙二醇法诱导烟草原生质体融合的条件优化

采用聚乙二醇(PEG)法诱导烟草原生质体融合,对融合时PEG的浓度、用量、融合时间及融合温度进行优化,以期得到一种融合率较高的方法。结果表明:以40%的PEG融合的二元融合率最高;PEG与原生质体悬浮液体积比为3∶5,在0.5～0.6 mol/L甘露醇为渗透压时,25～30℃下融合20～25 min的融合效果最佳,融合率始终保持在20%左右,甚至可达25%。

人防御素-1(HNP-1)在烟草原生质体中的瞬间表达

原生质体瞬间表达系统一般用于研究特定基因5′和3′端不同序列在相应基因表达中的作用、不同启动子对于特定基因的转录活性及为外源基因最终导入植株摸索实验条件,而将其用于研究动物基因在植物细胞中的表达情况则少见报道。

烟草花叶病毒影响烟草细胞活力的初步研究

以普通烟草原生质体为材料,初步研究了烟草花叶病毒(TMV)对寄主细胞活力的影响。结果表明,在接种20 d内,TMV对寄主叶肉细胞的分裂有抑制作用,且对不同细胞的生活力有不同的影响,并通过原生质体的培养做了验证。

甘蔗和烟草幼叶原生质体的核酸含量与核酸酶活性及其影响因素

与幼叶组织相比，酶法新鲜分离的甘蔗和烟草幼叶原生质体内的ＲＮＡ、ＤＮＡ及总核酸含量均降低。其原因可能是刚游离的原生质体内酸性和碱性ＲＮＡ酶与ＤＮＡ酶等活性提高所致。甘蔗叶原生质体内的核酸降低量和ＲＮＡ酶与ＤＮＡ酶活性的增加程度均高于烟草。随用作渗透压稳定剂的甘露醇浓度增加，甘蔗和烟草叶原生质体的ＲＮＡ酶和ＤＮＡ酶活性均相应提高。其中以甘蔗叶原生质体的核酸酶活性增加水平较明显。在细胞壁降解产物的作用下，除了甘蔗原生质体内的ＲＮＡ酶活性略被促进外。

普通烟草与黄花烟草体细胞杂种选育出新品系

通过烟草原生质体融合获得普通烟草(N.tabacum)与黄花烟草(N.rustica)的种间体细胞杂种植株,用普通烟草回交后,在其后代中选择优良单株,经二年的产质和抗性鉴定,证明是一个遗传性状稳定、抗病的优良新品系,在烤烟生产上有一定的利用价值,使烟草体细胞杂交技术由实验阶段进入应用研究阶段。

美国烟草G140细胞工程育种初步研究

在美国烟草G140髓细胞离体培养再生植株过程中,筛选了几株叶片厚度、叶绿素含量、光合和呼吸都高于种子苗的变异组培苗,其中三株感染花叶病指数大大低于种子苗,组培变异株与种子苗的过氧化物酶同工酶有明显差异。G140烟草细胞脱壁进行原生质体培养形成再生植株。用叶圆片法将CAT(抗氯霉素基因)及细胞分裂素基因导入G140叶肉细胞并得到表达,形成小植株。