烟草愈伤组织中不被KCN加氧肟酸抑制的剩余呼吸研究

烟草愈伤组织中存在有大约占总呼吸20—30%的不被 KCN 加 m-CLAM 抑制的“剩余呼吸”,它的性质和在细胞中的定位都不清楚。本研究发现,乙醇酸和乙醛酸都明显促进不被KCN(或 NaN_3)加 m-CLAM 抑制的剩余呼吸;乙醇酸听引起的氧吸收和剩余呼吸可被α-羟基乙磺酸钠抑制;线粒体呼吸被 KCN 加 m-CLAM 完全抑制;除去线粒体的上清液中发现有乙醇酸和乙醛酸氧化活性的存在。初步确定,烟草愈伤组织中这部分剩余呼吸主要是由非线粒体乙醇酸氧化酶所催化的。     

Effects of KCN and NaN3 Pretreatment on the Cyanide-Resistant Respiration in Tobacco Callus

The effects of KCN (0.5mmol/L) and NaN3 (0.01 mmol/L) pretreatment on the operation of the alternative pathway in subcultured tobacco (Nicotiana rustica L. cv. Gansu yellow flower) callus were analyzed. After treatment with KCN and NaN3 for 12 h, the total respiration rate (Vt) decreased by 12% and 17%, whereas oxygen consuption by the cytochrome pathway decreased by 22% and 28% respectively. The capacity of the alternative pathway (Valt) remained constant, while the activity of the alternative pathway (ρ· Valt ) inreased slightly. This changing pattern led to a declined contribution of the cytochrome pathway to the total respiration rate and an increased activity of the alternative pathway. Treatment with KCN for 24 h brought about a slight rise of oxygen consumption by the cytochrome pathway as compared with that in callus treated for 12 h, but the oxygen consumption was still lower than that in the untreated callus. Treatment with NaN3 for 24 h resulted in a profound decrease of the cytochrome pathway operation and a continuing increase of the alternative pathway operation. These data indicated that the enhanced operation of the alternative pathway played a compensatory role to the total respiration when the cytochrome pathway was partially inhibited in tobacco callus.     

Studies on the CN-resistant Respiration in Callus of Nicotiana rustica,Gansu Yellow Flower

In attempting to examine whether CN-resistant respiratory pathway is present in callus culture, we used tobacco callus cultures grown on different media. The M-1 medium contained tbe mineral and organic elements of MS medium and was supple,nented with 6-BA (0.5 mg/l) and 2,4-D (2 mg/l), and M-2 medium with 6-BA (2mg/l) and IAi (1 mg/l). No differentiation was observed in both of them. The respiration of M-1 callus was partly resistant to CN, and was markedly inhlbited by m-CLAM in the presence or absence of CN. Experiments of m-CLAM titration showed that the averages of relative contribution of alternative and cytochrome pathway in M-1 callus were 31% awl 46%of the total respiration respectively during the euliure period of 25 days. A same experiment was made on the M-2 callus. It was found that the pereeutages of relative contributions of the two electron transport pathways to the total respiration were approximately the same as those of the M-1 callus, although the respiratory rate was higher in M-2 callus. The above results showed that the bulk of respiratory electron flux was mediated by the eytoehrome pathway, although the alternative pathway was operative in callus of tobacco. The change of exogenous hormones added in the medium could not produee significant effects on the degree of relative contribution of two electron transport pathways under non-differentiation conditions.     

Changes of the CN-resistant Respiration in Callus Cultures of Nicotiana tabacurn during the Period of Differentiation

The callus cultures obtained from unpollinated young ovaries of willow leaf tobacco were transplanted separately on subculture medium (M-l) and differentiation medium (M-2). They were called M-1 callus and M-2 callus respectively. Only meristematic cells and parenchymas could be observed in M-1 callus. The M-2 callus, however, was able to differentiation into vascular systems, embryoids and bud primordia. The respiration of M-1 callus exhibited CN-insensitive. The relative contributions of alternative pathway and eytochrome pathway to the total respiration were 29–38% and 44–51% respectively during the culture period of 25 days. It was obvious that the bulk of the respiratory electron flux was mediated by the eytochrome pathway. The greater part of the respiration in M-2 callus was insensitive to cyanide. The relative contributions of alternative pathway and cytochrome pathway to the total respiration were 41–47% and 29–32% respectively. These results suggested that the increase in the participation of the alternative pathway in total respiration was probablely related to the differentiation of the callus culture.     

烟草愈伤组织在热胁迫过程中活性氧与抗氰呼吸变化的关系

烟草愈伤组织在 0～ 14h高温 (4 0℃ )胁迫期间 ,其体内的O- ·2 和H2 O2 迅速积累 ,分别在 2和 6h左右达到极大值。同时清除它们的酶SOD和CAT活性迅速上升 ,极大值的分布位置分别为 2和 10h左右 ,而胁迫期间的抗氰呼吸比未胁迫 (0h)时显著地下降 ,但在胁迫期间抗氰呼吸于 6h左右出现一峰值 ;用外源H2 O2 和O- ·2 及清除它们的酶的抑制剂AT和DCC分别处理愈伤组织 ,发现抗氰呼吸明显地加强。推测交替氧化酶可能同抗氧化酶如SOD和CAT一样参与了活性氧的清除 ,提出活性氧含量的变化可能是导致热胁迫过程中抗氰呼吸变化的主要原因之一。     

Studies on 6-benzyladenine Localization in Callus Cells of Tobacco

The localization of 6-benzyladenine in the callus cell of tobacco was studied by means of autoradiography. Electron microscope autoradiographs showed that the silver grains localized at not only cell membrane system such as plasma membrane, tonoplast, plastid membrane and mitochondrion membrane but also nucleus especially nucleolus. The results of the present study indicated that 6-benzyladenine receptor site associated with both cell membrane system and nucleus.     

Effect of Cytokinin on the Enhancement of Nitrate Reductase NR Activity in Tobacco Callus Tissues and Wheat Seedlings

The effect of cytokinin on the formation of NR activity were studied with tobacco callus tissues and wheat seedlings. Cytokinin could not induce the NR activity alone but could enhance the NR inducibility (Table 1). The enhancement of NR formation was detected in the tissues pretreated with cytokinin for over 12 hours. It showed that there was a precondition in the tissues for the induction of NR (Fig. 3). The precondition could not be improved by cytokinin when cycloheximide (inhibitor of protein synthesis) was added into the medium during cytokinin pretreatment (Table 2). Thus, it was thought that cytokinin might enhance synthesis of a protein which participated in the NR activity induction. In immunological test (Fig. 5) the existence of a nonactive apoenzyme of NR in higher plant tissues was demonstrated. It is, therefore, suggested that there are two major steps in the NR activity formation: (l) the synthesis of a nonactive NR apoenzyme, (2) the activation of this nonactive apoenzyme. The former step might be stimulated by cytokinin and the latter was mediated by nitrate.     

Studies on the Salicylic Acid Inducted Lipid Peroxidation and Defense Gene Expression in Tobacco Cell Culture

Salicylic acid (SA) could inhibit catalase activity, induce rapid lipid peroxidation and PR-1 gene expression of the tobacco ( Nicotiana tabacum L. ) cell culture which was incubated with exogenous SA. Ρ-ihydroxybenzene and H2O2 could also induce lipid peroxidation and PR-1 gene expression at different level, but they were not able to inhibit the catalase activity of tobacco cells. Inhi0itors of mRNA and protein-synthesis (a-amanitine and cycloheximide, respectively) could not induce both lipid peroxidation and PR-1 gene expression of tobacco cell culture. However, coordinated action with SA respectively, a-amanitine or cycloheximide was able to induce lipid peroxidation effectively, but strongly blocked the activation of PR-1 gene expression by SA in tobacco cell culture. These results suggested that the generation of reactive metabolites or free radicals, which were induced by SA or other inducers through reaction with catalase or other compounds, initiated lipid peroxidation, subsequently activated pathogen-resistance genes expression. Obviously the lipid peroxidation molecule played an important role in SA signal transduction in tobacco.     

咖啡酸和氯化钴对茉莉酸甲酯诱导抗病相关酶活性的影响

烟草愈伤组织在含咖啡酸(5mmol/L)和／或CoCl2(10mmol/L,乙烯合成抑制剂)的MS培养基上暗培养,同时用莱莉酸甲酯(1mg／d,简称MJ)处理愈伤组织。处理后测定乙烯、水杨酸和病程相关蛋白(PR)含量及一些抗病相关酶活性。MJ明显促进乙烯产生、增加水杨酸和PR蛋白含量,提高苯丙氨酸解氨酶(PAL)、β1,3—葡聚糖苷酶和几丁酶的活性。咖啡酸降低MJ对乙烯和水杨酸诱导,CoCl2明显降低MJ对乙烯的诱导,但没有明显影响MJ对水杨酸的诱导,两者都促进MJ诱导PAL活性而抑制MJ诱导β1,3—葡聚糖苷酶活性。咖啡酸明显影响MJ诱导内切几丁酶,几乎完全抑制对外切几丁酶的诱导;CoCl2对MJ诱导内切几丁酶没有影响,促进对外切几丁酶的诱导。实验结果表明,不同的抗病相关酶活性诱导有不同的信号传递途径,在所测几种酶的诱导中,水杨酸起主要作用,乙烯作用较小,MJ的诱导作用主要是由水杨酸所转导。     

Possible Participation of Active Oxygen Species in the Induction of Cyanide-Resistant Pathway at the Initial Senescence of Tobacco Callus

Changes in active oxygen species (AOS) and respiration, with special reference to the alternative pathway of respiration, were followed in callus of tobacco (Nicotiana rusticaL.) during its growth and senescence. Two peaks of the total respiration rate were observed: the first one appeared on day 11, and the second one on day 19 (in senescing callus). H₂O₂and O^– ₂contents increased gradually and reached the greatest values when callus senescence initiated (by day 11 and day 15, respectively), and then declined. The peaks of H₂O₂and O^– ₂before the onset of senescence coincided with the peaks of capacity V _altand activity V _altof alternative pathway, respectively. After the onset of senescence, ·OH accumulated abundantly and maintained at a relatively high level from then on, accompanied by the decrease in both V _altand V _alt. The conclusion is that these three active oxygen species operated coordinately to regulate the alternative pathway during growth and senescence of tobacco callus, as confirmed by the callus treatments with AOS scavengers, exogenous ·OH, and inhibitors of antioxidant enzymes. The possibility of AOS-induced alternative pathway respiration at the initial senescence of tobacco callus is discussed.     

顶空气相色谱法测定熊去氧胆酸中3种有机溶剂残留量

目的:建立顶空气相色谱法同时测定熊去氧胆酸原料药中3种有机溶剂残留量的方法。方法:用0.25 mol·L-1的氢氧化钠溶液溶解样品,采用顶空进样-气相色谱法,应用FID检测器,在DB-WAX毛细管色谱柱(30.0 m×320μm×1.0μm)上程序升温(起始温度70℃,保持5分钟,再以15℃·min-1的升温速率升至180℃,保持5分钟),载气为氮气,进样口温度200℃,检测器温度230℃。结果:待测组分均能得到有效分离,各组分在所考察的浓度范围内线性关系良好,r=0.9976~0.9996,平均回收率为91.00%~99.09%。结论:本方法可准确测定熊去氧胆酸原料药中丙酮、乙醇及N,N-二甲基甲酰胺的残留量,操作简便、准确度和灵敏度高,可达到有效控制其质量的目的。     

不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较

比较了不同低温(14℃和4℃)胁迫对烟草(Nicotiana rusticaL．)愈伤组织抗氰交替途径诱导和交替氧化酶表达的影响。结果显示,不同低温胁迫处理能显著诱导烟草愈伤组织交替途径容量和实际运行的增加,且都呈现出基本相同的变化模式：在胁迫的初期(1—3d)持续增加,在3d时达到最高,而后下降到一个相对恒定的水平。但交替途径容量增加的幅度与温度下降的程度密切相关,而交替途径实际运行量的诱导程度在不同低温胁迫下的差异却很小。表明交替途径容量和实际运行对低温胁迫的响应是不同的。免疫印迹分析结果表明：低温胁迫明显诱导了交替氧化酶总蛋白的增加,且其随低温胁迫进程的变化与交替途径容量的变化基本一致;而对交替氧化酶单体与二聚体在低温胁迫下的含量变化检测结果则显示,烟草愈伤组织中交替氧化酶主要以二聚体形式存在,且这一存在形式并不随低温胁迫程度的加深而发生改变。两种形式的交替氧化酶蛋白含量都能被低温胁迫诱导增加,但其单体水平在两种不同的低温胁迫下并无明显差别,而4℃低温胁迫诱导的二聚体交替氧化酶蛋白含量明显高于14℃。表明不同程度低温对抗氰交替途径发生的不同影响主要是由于对交替氧化酶蛋白二聚体形式的不同诱导程度所致;而高活性的交替氧化酶单体形式则不因低温胁迫程度的加重而被明显诱导升高,使得抗氰交替途径的运行程度在两种不同的低温胁迫处理条件下无显著差异。     

单克隆抗体和抗合成多肽抗体检测烟草愈伤组织中交替氧化酶表达的比较

单克隆抗体和抗合成多肽抗体检测烟草愈伤组织中交替氧化酶表达的比较 晏婴才 林宏辉* 梁厚果 杜林方 （四川大学生命科学学院,成都610064I）     

无患子愈伤组织诱导的多因子正交试验研究

目的:探寻无患子愈伤组织诱导的最佳外植体、培养基和培养条件。方法:采用5因素混合水平正交设计,用DPS2000统计软件对其结果进行方差分析和多重比较(SSR)。结果:诱导愈伤组织的最佳外植体为当年生枝条水培芽的嫩茎段,用0.2%HgCl2消毒2min,其无菌存活率为98.33%,培养条件为光照或黑暗,最佳培养基配方为:MS(B5) 6-BA 1.5mg.l-1 NAA0.2mg.l-1,其愈伤组织的诱导率为97.78%。结论:激素配比是影响愈伤组织诱导的主要因素。较高的6-BA浓度(1.5~2.5mg.l-1)与较低的NAA浓度(0.2mg.l-1)配比,有利于无患子愈伤组织的诱导。     

不同理化因子对雪莲培养细胞中黄酮类形成的影响

研究了不同理化因子对水母雪莲(Saussurea medusa Maxim)愈伤组织生长及黄酮类化合物生物合成的影响。结果表明,有利于细胞生长及黄酮形成的合适温度为25℃。白光对愈伤组织生长无促进作用,但有利于黄酮的形成。培养基中添加1mg／L NAA和O．2mg／L的KT组合对细胞的生长较有促进作用。5％蔗糖和1％葡萄糖的组合有利于细胞的生长和黄酮的形成。用⁶⁰C0-γ射线辐照愈伤组织,在剂量为4000Gy的条件下,获得一个合成黄酮能力高于原愈伤组织70％的细胞系。用高效液相和紫外分光光度法,测定离体培养光照条件下干细胞总黄酮的含量为3．2％,是暗培养的4．4倍。培养温度25℃时干细胞黄酮的含量为2．02％,分别为20℃,35℃时的5倍和3．2倍。     

High Rabdosiin and Rosmarinic Acid Production in Eritrichium sericeum Callus Cultures and the Effect of the Calli on Masugi-Nephritis in Rats

During an investigation of plant cell cultures that might be useful in the treatment of renal disorders, we established a vigorously-growing E-4 callus culture of Eritrichium sericeum that produced large amounts of caffeic acid metabolites, (?)-rabdosiin (1.8% dry wt) and rosmarinic acid (4.6% dry wt). Elicitation of the calli by methyl jasmonate induced a 38% increase in total polyphenol production. The most efficient method of eliciting (?)-rabdosiin biosynthesis was through the treatment of E-4 calli with cuprum glycerate, which induced an increase in (?)-rabdosiin production of as much as 4.1% dry wt. Oral administration of E-4 callus biomass (100 mg/kg/d for 30 d) to rats with induced Masugi-nephritis caused an increase in diuresis and lowered creatinine excretion and proteinuria levels as compared with Masugi-nephritis untreated rats. While all of the Masugi-nephritis untreated rats began to suffer, near a quarter of the E-4 treated rats remained in good health. This result indicates that the E-4 culture has the potential to alleviate the symptoms associated with nephritis.     

Effect of Ultraviolet (UV-B) Radiation on the Formation and Localization of Phenolic Compounds in Tea Plant Callus Cultures

The effect of ultraviolet (UV-B) radiation on the accumulation and tissue localization of phenolic compounds in two strains of callus cultures of tea plant (Camellia sinensisL.) were investigated. The strains differed in their morphological and physiological characteristics and biosynthetic capacity. UV-B radiation hampered culture growth, decreased the size of callus-forming cells and promoted the accumulation of soluble and, to a lesser extent, polymeric forms of phenolic compounds, such as lignin. This accumulation was accompanied by an increase in the phenolic compound deposition in cell walls and intercellular space and by deposition of a lignin-like material on the surface of callus cultures. The strain characterized by an increased formation of phenolic compounds was more resistant to UV-B radiation as compared to that with lower phenolic productivity.     

Effects of Exogenous Indole Butyric Acid and Callus Formation on the Anti-oxidant Activity, Total Phenolic, and Anthocyanin Constituents of Mulberry Cuttings

In order to evaluate the effects of exogenous indole butyric acid （IBA） and callus formation on the antioxidant activity, total phenolics, and anthocyanin constituents of Morus nigra L. and M. alba L. cuttings, we investigated the variations before and after the treatment. The results indicate that anti-oxidant ability, total phenolic, and anthocyanin constituents of the callus stems of both Morus species were higher than those of non-callus forming species. There were also increases observed in anti-oxidant ability, total phenolic, and anthocyanin constituents of calli treated with IBA （1 000-3 000 mg/L）.     

安徽烟草病毒类型及优势种初步研究

1978年,烟草病毒病在安徽烟区流行,导致凤阳县烟叶总产损失92.3％,引起了普遍震惊。1981—1984年作者对来自16个县、市552个病毒材料,经生物测定、血清反应、电镜观察,初步分离出CMV、TMV、PVY和PVX四种病毒。它们分别约占检测总数的82.79％、4.53％、2.54％和0.36％,其中CMV与长期视为优势种的TMV比值为18.3,除此,尚有约占检测总数9.8％的CMV和TMV、CMV和PVY复合侵染,以及不明类型的毒株。通过对田间烟草以及其他植物花叶病株的实际检测,进一步表明:CMV在烟区分布范围极广、出现频次最多,已形成了复杂的循环侵染系统,成为近期内烟草病毒病持续流行危害的首要毒原。