烟草品种RAPD分子标记遗传差异研究

用２３５个随机引物对来源于中国,美国等国家的２３个烤烟和地方晒晾烟品种基因组ＤＮＡ进行了ＲＡＰＤ分析。结果表明：２５个引物可以扩增出多态性产物;利用其中１６个引物共扩增出１２８条带,其中４６条品种间表现多态性,这种多态性可以进行品种鉴定;根据多态性的带计算了品种间遗传距离,按类平均法可将２３个品种划分６类,各类内含不 地理来源和不同调制类型的品种,说明品种地理来源和调制类型差异与遗传差异没有必然的     

烟草NtMTPC4启动子的克隆与缺失分析

组织特异性启动子是基因工程研究及实际应用中的重要工具。为了研究烟草NtMTPC4启动子的表达特性,采用实时荧光定量PCR方法分析烟草金属耐受蛋白C4(MTPC4)基因NtMTPC4在烟草不同组织中的表达模式,并利用PCR技术克隆得到NtMTPC4的上游启动子NtMTPC4-P。根据调控元件的分布对NtMTPC4-P进行不同程度的缺失,获得了NtMTPC4-P1、NtMTPC4-P2和NtMTPC4-P3缺失体;构建全长NtMTPC4-P和不同缺失体的植物表达载体,并通过花序浸染法转化拟南芥,对获得的转基因拟南芥进行GUS组织化学染色分析。结果表明,NtMTPC4基因的表达具有组织特异性,其在花中的表达量最高。NtMTPC4-P全长2 287 bp,含有CAAT-box、TATA-box、GTGANTG10和POLLEN1LELAT52等顺式作用元件和调控元件。GUS组织化学染色结果表明,全长启动子NtMTPC4-P和不同缺失体都能特异性地驱动GUS基因在转基因拟南芥花粉中的表达。本研究结果为通过基因工程技术在花粉中特异表达目的基因提供了新的调控元件。     

Some Responses of Two Nicotiana tabacum L. Cultivars Exposed to Vanadium

The effect of exposure to different vanadium (V) concentrations was studied in Nicotiana tabacum L. (cv. ‘Burley’ and cv. ‘Virginia’). In both cultivars, the vanadium did not affect the percentage of germination but root and shoot growth was inhibited, especially with the 80 μM dose. An altered root morphogenesis in vanadium growth plants was observed at the end of the experiment. The highest V accumulation was found in roots and the results showed a direct correlation between V content and V supply (P?≤?0.01). The V concentrations did not modify nicotine content in the cv. ‘Burley’, but in the cv. ‘Virginia’ the highest nicotine content was found in vanadium exposed plants (P?≤?0.05). The results suggest that these V concentrations may have a possible elicitor effect on alkaloid synthesis in N. tabacum L. cv. ‘Virginia’, and that this cultivar has a higher V sensitivity than the cv. ‘Burley’.     

Silicon alleviates long-term copper toxicity and influences gene expression in Nicotiana tabacum

Silicon (Si) is beneficial for plant growth and aids in stress tolerance. In this study, the effects of Si on long-term copper (Cu) toxicity in the low Si accumulator Nicotiana tabacum were evaluated. Silicon supplementation alleviated growth inhibition in roots and shoots of N. tabacum exposed to Cu toxicity. Alleviation of Cu toxicity correlated with increased Si accumulation in roots and leaves, suggesting N. tabacum contains a stress-regulated mechanism for Si transport. Root Cu concentration decreased in Si-supplemented plants exposed to Cu toxicity. Interestingly, Copper Transporter 1 (COPT1) expression decreased in roots of Si-supplemented plants exposed to Cu toxicity, which may contribute to Cu uptake reduction. Decreases in ethylene (ET) biosynthetic gene expression were previously implicated in Si-mediated stress alleviation. In the present study, Si-mediated alleviation of Cu toxicity corresponded with increased ET biosynthetic gene expression.     

烟草染色体倍性快速鉴定方法

摘要: 对两个杂交组合(G28 × NC2326和K326 × Coker176) F1烟草 (Nicotiana tabacum) 花药培养诱导出763个不同倍性植株。基于植株的花和种子结实率常规鉴定倍性水平比较，用气孔保卫细胞叶绿体数目预测植株倍性准确率达93.52%。表明采用气孔保卫细胞叶绿体计数法可以在苗期快速、准确地确定植株染色体倍性。同时, 在移栽前间接地鉴定、筛选出叶绿体数<14的单倍体苗，作进一步秋水仙碱加倍处理，以减少对单倍体材料的浪费，加快DH群体构建速度。     

草甘膦对烟草叶片超微结构的影响

草甘膦是农业生产中广泛使用的广谱非选择性除草剂,它抑制5-烯醇丙酮酸莽草酸-3-磷酸合成酶。该酶是莽草酸途径中的关键酶,广泛存在于微生物与植物中。用30mmol／L草甘膦处理烟草叶片2、6、12、24、48h,结果显示,处理12h内烟草叶片无明显变化,24、48h后叶片黄化,并出现坏死。对各处理时期烟草叶片的超微结构研究显示,草甘膦处理导致烟草叶片细胞的叶绿体基粒片层结构被破坏,线粒体电子密度下降。这些结果提示,草甘膦通过阻碍植物的光命作用和呼晖作用面导致叶片坏死。     

Identification of fructo- and malto-oligosaccharides in cured tobacco leaves (Nicotiana tabacum)

Fructo-oligosaccharides (FOSs) and malto-oligosaccharides (MOSs) in cured tobacco leaves ( Nicotiana tabacum ) were detected and quantified using high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). These oligosaccharides were present in several tobacco materials including flue-cured tobacco, sun/air-cured tobacco, and cut filler of commercially available tobacco products, but were not detected in air-cured tobacco. The changes in these oligosaccharides during storage were also investigated. The results revealed that MOSs simply decreased under a warm atmosphere and FOSs increased. In addition, the formation of FOSs in cured tobacco leaves occurred in the presence of sucrose. With regard to FOSs, it has been reported that green tobacco leaves do not contain FOSs such as kestose, nystose, and fructosyl-nystose. The results of a model test suggested that the changes in FOS amount were caused by enzymatic reactions.     

转PSAG12-BAS1基因抑制烟草叶片的衰老

衰老被认为是烟草(icotiana tabac um)叶片发育的最后阶段,伴随着叶绿素,脂类等降解,严重影响了光合产物的积累,导致作物产量降低、品质下降.因此,利用转基因技术使烟草在生长期间衰老延迟,光合产物量增加,从而增加产量,同时在收获之后,延缓衰老的叶片,可以维持烟草的新鲜程度,解决储存及运输问题.本研究利用农杆菌(Agrobacterium tumefaciens)介导的遗传转化法将含有衰老相关基因12 (senescence-associated gene 12,SAG12)启动子驱动光敏色素B激活标签的抑制蛋白1(phyB activationtagged suppressor1,BAS1)基因表达的元件导入烟草中,经抗性筛选和PCR鉴定,共获得45株转基因植株,其中有8株转基因烟草叶片具有衰老延缓现象.在叶片开始衰老时对野生型和转BAS1基因烟草叶片叶绿素含量、保护酶活性检测以及植物生长期状态进行观察测定,结果表明,转BAS1烟草植株叶绿素含量从顶端到基部均高于野生型;野生型和转BAS1基因烟草超氧化物歧化酶(super-oxide dismutase,SOD)活性和丙二醛(malondialdehyde,MDA)含量分析表明,转BAS1烟草植株中SOD活性比野生型高了31.98％,MDA含量比野生型降低了48.28％,通过对烟草生长发育过程观察,转基因烟草比野生型烟草衰老延缓10～15 d.在烟草叶片开始衰老时测定野生型和转BAS1基因植物细胞分裂素含量,结果发现,野生型烟草细胞分裂素的含量比转基因烟草降低了40.2％,上述结果说明,转BAS1基因延缓了烟草叶片的衰老,与细胞分裂素含量、保护性酶活性提高以及衰老性启动子启动有关.本研究为进一步研究SAG12-BAS1基因功能机制提供理论依据,同时该基因为创制转基因抗衰老材料提供了基础.     

Comparing Application Methods for Boron Fertilizer on the Yield and Quality of Tobacco (Nicotiana tabacum L.)

The present study is based on the hypothesis that different methods of supplying boron (B) may have different effects on the yield and quality performance of tobacco crop. A field experiment was conducted to study the effect of different methods of B fertilization on the yield, quality, and leaf composition of flue-cured Virginia tobacco (cv. K-399) in 2007 at the Pakistan Tobacco Research Station, Mansehra. Three different methods (i.e., foliar spray, soil application, and root dipping at the rates of 0.25 kg ha^–1, 1 kg ha^–1, and 0.5 mg B L^–1, respectively) in the form of boric acid along with a control (where no B was applied) were used in a randomized complete block design and replicated four times. Results revealed that different methods of B application significantly affected tobacco yield, quality, and nutrient uptake compared to the control. Maximum leaf area of 707 cm², green leaf yield of 18553 kg ha^–1, cured leaf yield of 2314 kg ha^–1, grade index of 79%, nicotine content of 2.54%, and sugar content of 18.35% were noted in the treatment plot where B was applied as a foliar spray; however, in a few cases, there was no significant difference found among different methods of B application. Chloride and potassium contents were not significantly affected by any method of B fertilization. The B fertilization increased the concentration of this element in tobacco leaf, and a maximum concentration of B 48.55 mg kg^–1 was noted in the foliar spray treatment. Moreover, fertilizer-use efficiency for different methods of B application revealed that foliar spray is more efficient as compared to soil application and root-dipping methods. Nutrient ratios to B such as potassium (K) / B and chloride (Cl) / B considerably decreased in tobacco leaf with increases in the concentration of B in leaves. These ratios provided some indication of the interrelationship of B with these nutrients in tobacco plants. Results also revealed that K/B and Cl/B ratios closely correlated with the grade index of tobacco leaf, and good grades of tobacco were found to be at a K/B ratio of 682 and a Cl/B ratio of 148 under the experimental conditions. The overall results indicated that the B foliar spray at the rate of 0.25 kg ha^–1 significantly increased the yield, quality, and the nutrient uptake by the tobacco crop under the prevailing conditions and was more effective than other methods of B application.     

Exogenous proline mitigates the inhibition of growth of Nicotiana tabacum cultured cells under saline conditions

The addition of exogenous proline (10 mm) to Na100-saline culture medium, modified LS medium (Linsmaier and Skoog 1965: Physiol. Plant., 18, 100–127) with 100 mm NaCl promoted the growth of tobacco (Nicotiana tabacum L., cv. Bright Yellow-2) suspension cells unadapted to salt stress without maintaining a high ratio of K⁺ to Na⁺ ions under salinity conditions. The addition of exogenous glutamic acid or alanine were not comparable to that of exogenous proline. The proline contents of the NaCl-unadapted cells became much higher when the cells were grown in Na100-saline culture medium with 10 mm proline than when the cells were cultured without proline. The accumulation of K⁺, Na⁺, counter ions was sufficient to compensate for the increase of the water potential of the cells caused by salinity. These results suggest that exogenous proline does not act as a nitrogen store and that proline may act as a protectant for enzymes and membranes against salt inactivation rather than as a compatible solute in tobacco suspension cells.     

Physiological Responses of Transgenic merA-TOBACCO (Nicotiana tabacum) to Foliar and Root Mercury Exposure

Plants expressing a modified bacterial mercury reductase, merA, are highly resistant to Hg(II) toxicity as a result of the enzymatically catalyzed electrochemical reduction of Hg(II) to the much less toxic and volatile Hg(0). merA expression may allow plants to manifest a suite of responses to mercury exposure, making them more capable than wild-type plants of interacting with and removing mercury from contaminated soil or water. We have engineered merA-expressing Nicotiana tabacum (tobacco) as a model plant for examining these responses. Mercury resistance was demonstrated by germinating and growing merA tobacco seeds on semi-solid medium spiked with a HgCl₂ concentration acutely toxic to wild-type plants. On similar growth medium, merA plant roots penetrated a highly concentrated, localized Hg(II) zone of HgS (cinnibar) more readily than wild-type roots. In hydroponic medium spiked with HgCl₂, merA plants maintained higher evapotranspiration activity than wild-type plants. The ability of merA Hg(II)-reductive activity to counter typical plant-catalyzed Hg(0) oxidation to Hg(II) was demonstrated by a lower net foliar absorption of atmospheric Hg(0) than wild-type plants. Mercury translocation through merA plants was examined through reciprocally grafted merA and wild-type tobacco grown on HgCl₂-spiked hydroponic medium. Elevated mercury concentrations in wild-type shoots grafted to merA roots suggest the vertical movement of mercury within merA tissues or plants may be facilitated by dynamic balance between native Hg(0) oxidation and MerA-catalyzed Hg(II) reduction. These experiments demonstrate that merA-engineered tobacco plants display an array of tissue-level and whole-plant attributes which should allow for more efficient mercury extraction and processing compared to the wild-type.     

Identification of NtNIP2;1: an Lsi1 silicon transporter in Nicotiana tabacum

Foliar silicon (Si) concentrations vary significantly among plants, resulting in the classification of different species as either high or low-Si accumulators. While the uptake pathways have been identified in some high accumulators, those that operate in low accumulators have largely been overlooked. Using a bioinformatics approach, a putative NIP2-like Si transporter gene was discovered within the genome of the low-Si accumulator, Nicotiana tabacum. The predicted protein, termed NtNIP2;1, possesses all the molecular signatures expected of an NIP2 Si transporter. Furthermore, when NtNIP2;1 was expressed in Xenopus oocytes, Si transport across the plasma membrane was observed. Nicotiana tabacum NIP2;1 RNA levels in the roots of seedlings were down-regulated in response to Si, similar to the Si transporter, OsLsi1, in rice. Taken together, these data suggest that a functional NIP2 Si uptake mechanism is also present in plants that acquire low foliar Si levels.     

Transformation and Bioavailability of Selenate and Selenite Added to a Nicotiana tabacum L. Planting Soil

Selenium (Se) is an essential nutrient for humans and is beneficial for plant growth. To investigate the transformation and bioavailability of Se in tobacco planting soil, selenite and selenate were applied. A pot experiment and sequential extraction scheme were used to investigate the Se contents in different forms in soils treated with Se. A series of equations were applied to model the transformation behavior of Se in this study. The results showed that the forms of selenium were increased significantly by applying the different valence state of water-soluble selenium. The carbonate-bound and iron-manganese (Fe-Mn) oxide–bound species were improved in selenite-added soil, whereas the soluble and exchangeable forms were increased in selenate-added soil. Michaelis-Menten equation fitting results indicated that estimated maximal selenium contents of leaves, stems, and roots in selenate-added soils were 1.83, 15.81, and 20.98 times larger than in selenite-added soils. The utilization levels of selenate were 4.3 to 7.9 times larger than selenite for Nicotiana tabacum L. In conclusion, the bioavailability and mobility of selenate were greater than selenite in Nicotiana tabacum L. planting soil.     

Effects of arbuscular mycorrhizal inoculation on cadmium accumulation by different tobacco (Nicotiana tabacum L.) types

The effect of arbuscular mycorrhiza (AM) on cadmium (Cd) uptake by tobacco (Nicotiana tabacum L.) was studied in a pot experiment. Three commercial varieties, Basma BEK, K326 and TN90, representing three distinct tobacco types, were each grown in a different soil with nutritional conditions matching as closely as possible their requirements for field production. Cd concentrations in these soils were within the background range. Each variety was either non-mycorrhizal or inoculated with one of five AM fungal isolates. Cd concentration in leaves was decreased by inoculation with selected isolates in the K326 and TN90 variety grown in acidic soils. In contrast, it was increased by inoculation with most isolates in the Basma BEK variety grown in a basic soil with low Cd availability. Besides, plants of all three varieties had significantly higher leaf concentrations of phosphorus and nitrogen in some inoculated treatments. The percentage of root colonisation was mostly low in the inoculated treatments. In the Basma BEK and TN90 variety, the tested AM fungal isolates differed in their ability to colonise roots, but no correlation was found between the root colonisation of an isolate and its effects on the Cd concentrations in tobacco leaves. One isolate influenced most pronouncedly Cd concentrations and improved mineral nutrition in all the three combinations of variety and soil despite its low colonisation levels. AM symbiosis probably affected Cd uptake of tobacco by indirect mechanisms such as stimulation of root growth or mycorrhizal plant mediated changes in chemical or biological soil properties.     

烟草与丛枝菌根真菌的共生效应研究进展

丛枝菌根(Arbuscular mycorrhiza,AM)真菌是陆地生态系统中广泛存在的一类专性共生土壤微生物,是根系土壤区域中重要的功能菌群之一.AM真菌可侵染植物根系形成丛枝菌根共生体,改变植物根系形态和改善营养状况,从而提高宿主植物的生长发育、产量、质量和抗逆性.目前从烟草根系土壤分离报道的AM真菌已达13属5...     

Sucrose transporter NtSUT1 confers aluminum tolerance on cultured cells of tobacco (Nicotiana tabacum L.)

The role of plasma membrane-localized sucrose transporter (NtSUT1) was investigated using cultured tobacco cell (Nicotiana tabacum L.) line BY-2. The wild type (WT) cells were first transformed with the NtSUT1 gene or its fragments cloned from tobacco cell line SL to form the over-expression (OX) and suppression (RNAi) cell lines, respectively. Using OX and RNAi transgenics, the role of NtSUT1 in growth capacity of actively growing cells and in aluminum (Al)-treated cells was examined. During the logarithmic phase of growth in nutrient medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), both the rate of sucrose uptake measured with radio-tracer and the content of soluble sugars were higher in OX and lower in RNAi cell lines compared to WT. Overall, the content of soluble sugars negatively correlated with the time necessary for doubling mass (fresh weight). When cells were treated without (control) or with Al in a simple medium containing calcium, sucrose and 2-(N-morpholino)ethanesulfonic acid (MES; pH 5.0) for up to 18 h, the expression of NtSUT1 under its native promoter, or under the control of strong constitutive cauliflower mosaic virus (CaMV) 35S promoter, was strongly dependent on the presence of 2,4-D. Thereafter, the cells were preferentially treated in the presence of 2,4-D. During 6 h after a start of the control treatment, sucrose uptake rates were, compared to WT, slightly higher and lower in OX and RNAi lines respectively. The addition of Al reduced the sucrose uptake rates of OX and WT to the level of RNAi line, indicating that Al inhibits sucrose uptake via NtSUT1. During the post-Al culture of control and Al-treated cells in a nutrient medium, sucrose uptake rates were much higher in OX compared to WT and RNAi lines, which closely and positively correlated with the growth capacity of the cells. Judging from the growth capacity of Al-treated cells relative to that of control cells, OX cells were more tolerant to Al than WT and RNAi. In summary, we conclude that over-expression of NtSUT1 confers higher growth capacity in actively growing cells as well as in Al-treated cells.     

氮钾肥施用方法对烤烟品质及经济效益的影响

针对河南烟区烤烟生产中施肥时所提供的氮、钾量与烟株生长的需氮、钾规律不吻合现状,采用大田小区试验,设置不同烟草专用氮钾肥基追肥比例10∶0、7∶3、5∶5,并配合钾肥在烟株生长旺长期—圆顶期不同兑水淋施处理,研究不同氮钾肥施用方法对烤烟化学成分、中性致香成分质量分数及经济效益的影响。结果表明：1）采用前期施足基肥（70%烟草专用肥）,适量追肥（30%烟草专用肥）,硝酸钾在旺长期—圆顶期分3次兑水追施的施肥方式能够显著提高中、上部烟叶的钾质量分数,使烟叶糖/碱比、钾/氯比趋于协调,提高了上部烟叶的可用性;该处理与对照相比,中、上部烟叶钾质量分数分别提高了49.70%和95.2%。2）从香气物质上来看,该处理中部烟叶中性致香成分中的棕色化产物、类胡萝卜素类、新植二烯香气物质和香气总量分别比对照提高6.59、45.45、249.13和326.89μg/g,增幅分别达36.96%、36.29%、51.70%和49.52%。3）从经济效益上来看,该处理与对照相比增产125.05 kg/hm2,增加产值3 737.81元/hm2。因此,烟草专用肥以7∶3的比例分别在移栽时和大培土时施用、全部的硝酸钾肥在旺长期—圆顶期分3次兑水淋施的施肥措施下烟叶化学成分更协调。该处理增加了烟草香气物质的质量分数,从而显著提高了烟叶产值。