Low‐temperature derivatization followed by vortex‐assisted liquid–liquid microextraction for the analysis of polyamines in Nicotiana Tabacum

Polyamines are ubiquitous polycationic molecules that play a key role in many biological processes such as nucleic acid metabolism, protein synthesis, cell growth, and nicotine synthesis precursors. This work describes a rapid, sensitive, convenient, green, and cost‐effective method for the determination of polyamines in Nicotiana tabacum by ultra high performance liquid chromatography with photodiode array detection. The analytes were derivatized with 3,5‐dinitrobenzoyl chloride at low temperature (about 4°C) and then extracted with vortex‐assisted liquid–liquid microextraction. The experimental designs based on quarter‐fractional factorial design and Doehlert design were used to screen and optimize the important factors in microextraction process. Under the optimal conditions, the method was linear over 0.05–8.00 μg/mL with an r² ≥ 0.992 and exhibited good repeatability and reproducibility less than 6.0 and 6.9%, respectively. The limit of detection ranged between 0.013 and 0.029 μg/g. The newly developed method was successfully employed to analyze different leaf samples of Nicotiana tabacum, among which the polyamines contents were found to be very different. Moreover, tyramine, 1,3‐diaminopropane, homospermidine, and canavalmine were tentatively identified with the electrospray ionization quadrupole time‐of‐flight mass spectrometry. To our knowledge, this is the first report of identification of canavalmine in Nicotiana Tabacum.     

Cloning and functional analysis of chloroplast division gene NtFtsZ2-l in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2 . On the basis of the research on ftsZl family, we analyzed the function of NtFtsZ2-l gene in Nicotiana tabacum . Microscopic analysis of the sense and antisense NtFtsZ2-l transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-l gene is involved in plant chloroplast division.     

In situ localization of calmodulin mRNA in the developing tobacco anthers

The temporal and spatial expression patterns of calmodulin mRNA in the developing tobacco anthers were investigated by in situ RNA hybridization, using digoxigening-labeled anti-RNA probe. Calmodulin mRNA was distributed in various developmental stages of tobacco anthers, but the expression level had temporal and spatial differences distinctly. During early stage of anther development, the expression level of calmodulin mRNA was significantly high, mainly distributed in epidermis, tapetum and transfusion parenchyma cells and so on. Especially, more mRNA was accumulated in the nuclei and chromosomes of microspore mother cells prior to and during meiosis. With the development of anther, mRNA was decreased gradually in the anther wall and pollen. By mature pollen stage, only a stronger positive reaction still existed in the epidermis of anther wall and transfusion parenchyma cells. The results suggest that the temporal and spatial expression of calmodulin mRNA is closely correlated with cell division, pollen development and substance transport.     

Distribution of a kinesinrelated protein on Golgi apparatus of tobacco pollen tubes

The conventional kinesin and kinesin-related proteins (KRPs) constitute a large family of microtubule-based molecular motors. Members of this family bind to micro-tubules in an ATP-dependent manner. They all have microtubule-activated ATPaes activities, and play essential roles in the transport of various vesicles and organelles in eukaryotic cells[1―3]. The first plant kinesin-like protein was detected in tobacco pollen tube with an antibody against animal kinesin[4]. Recently, a number …     

烟草和元麦叶肉原生质体吸收尼古丁机制的研究

用 EA_3-867纤维素酶分离的烟草(Nicotiana tabacum)和元麦(Hordeum distichon)叶肉原生质体,分别保温在尼古丁溶液中,用紫外分光光度计测定它们对尼古丁的吸收。结果表明,这两种原生质体能迅速地吸收尼古丁;原生质体吸收尼古丁的量,随介质尼古丁浓度的升高而增多;在生理学pH范围内,介质的pH对原生质体吸收尼古丁没有多大的影响。初步认为,这两种原生质体对尼古丁的吸收是一种简单的扩散机制。     

Phosphodiesterase production in an aqueous two-phase system by Nicotiana tabacum 1507

Studies were conducted on the production of phosphodiesterases by Nicotiana tabacum 1507 cell suspension in an aqueous two-phase system formed by adding 4% polyethylene glycol (mol wt 20,000) and 7.5% dextran (mol wt 70,000) to the medium. The time course of growth, biosynthesis, secretion, and partitioning of phosphodiesterases was followed in comparison with N. tabacum 1507 cultivation as a free suspension. Partitioning of phosphodiesterases took place mainly in the bottom dextran phase, and a possibility was revealed for obtaining an enzyme preparation with high phosphodiesterase activity.     

转基因烟草中过量生长素对叶片生长发育的影响

分别构建了iaaM、iaaM和ipt共表达植物转化载体,农杆菌介导转化烟草,RT-PCR鉴定转基因株系,ELISA方法分析叶片IAA、IPA和ZR含量.结果表明:叶片IAA含量高低依次为株系iaaM10、iaaM_ipt3和野生型;而细胞分裂素含量高低依次为iaaM_ipt3、野生型和iaaM10.随着内源IAA水平升高,气孔密度减小、保卫细胞长度增加、栅栏组织细胞增大和叶片厚度增加.讨论了生长素和细胞分裂素含量对叶片细胞生长发育的影响.     

Determination of Terpenoids in Plant Leaves by GC-MS: Development of the Method and Application to Ocimum basilicum and Nicotiana langsdorffii

In this work a simple and sensitive analytical method was developed for the determination of terpenoids in plants using gas chromatography coupled to mass spectrometry (GC-MS). Three extraction methods were tested on Ocimum basilicum leaves to maximize terpene extraction: Steam Distillation, Ultrasound-Assisted Extraction, and Microwave-Assisted Extraction (MAE). Best results, both in number of identified compounds and in their concentration, were obtained with MAE, which was used in all subsequent analyses. In particular five terpenoids (α-pinene, β-pinene, limonene, linalool, and α-terpineol) were chosen for the quantitative determination, performed in Selected Ion Monitoring mode. Calibration curves using dodecane as internal standard were drawn, showing good correlation coefficients (0.9981–0.9993). All figures of merit were satisfactory; limits of detection were in the range 14–27 ng for all analytes. The method was also applied to some specimens of wild-type and transgenic (GR and rolC) Nicotiana langsdorffii. Plants were exposed to water and chemical stresses to evaluate possible effects on terpene content. Linalool was present in higher quantities in the plants GR and rolC; both stresses produce a decrease of its concentration if compared to the non-stressed plants. The concentration of α-terpineol, detected only in WT and GR plants, was higher for the genetically modified plant, substantially confirming the trend observed for linalool.