Evaluation of Transgenic <i>Nicotiana tabacum</i>with <i>deh</i>E Gene Using Transposon Based IRAP Markers

In the present study, five genetically modified herbicide tolerant Nicotiana tabacum cv. TAPM24 plants with a constructed vector pCAMBIA1301a carrying dehalogenase E (dehE) gene were compared with three non-transgenic controls using Tto1 retrotransposon specific IRAP markers. dehE gene was originally characterized in Rhizobium sp. and it produced an enzyme which degraded the Dalapon herbicide. IRAP protocol was applied on transgenic and non-transgenic plants to investigate the retrotransposon based genetic variation which may appear during transformation. Polymorphism rates were calculated as 0%-20% from IRAP-PCR products among all plant samples. These results show that transformation of tobacco plant with the dehE gene may cause Tto1 retrotransposon alterations appearing as different band profiles. The findings are expected to contribute to genetic engineering studies to obtain better results and also to understand how transposons contribute to features such as transgenesis. In our knowledge, this is one of the first experimental data of transgenic N. tabacum engineered with dehE gene originated Rhizobium sp. in terms of retrotranposon based variation.

Effect of Nitrogen Fertilization on Growth and Photosynthetic Nitrogen use Efficiency in Tobacco （Nicotiana tabacum L.）

Soil pot experiments were conducted in a greenhouse to examine the effects of different nitrogen （N） supply （low, 0.15 g N/kg; middle, 0.3 g N/kg; and high, 0.6 g N/kg dry soil） on the growth, photosynthetic characteristics and photosynthetic nitrogen use efficiency （PNUE） of tobacco seedlings （Nicotiana tabacum L. Yunyan 87）. The results showed middle and high N significantly enhanced seedling growth including plant stem and leaf dry weight comparing with low N. High N supply could lead to a dramatic increase in the photosynthetic capacity of tobacco seedlings under low N conditions. There were significant differences in leaf N content between nitrogen treatments. About a 76% increase in leafN content in plants fed by high N resulted in about 43% increase in Rubisco content and 27% in net photosynthetic rate. The non-corresponding increases in photosynthetic rate in tobacco seedlings fed by high N relative to low N resulted from Rubisco activity and/or carboxylation efficiency （CE）. These results indicated that tobacco seedlings under high N application can maintain high net photosynthetic rate （Pn） but lower PNUE, will finally result in a decline in N use efficiency.

Cloning and Expression of Calcium-Dependent Protein Kinase (CDPK) Gene Family in Common Tobacco (Nicotiana tabacum)

To further study the function of calcium-dependent protein kinase （CDPK） gene family in common tobacco （Nicotiana tabacum）, it is necessary to isolate more CDPKs from common tobacco and describe the sequence characteristics, evolutionary relationship and gene expression. Reverse transcription-PCR （RT-PCR）, rapid amplification of cDNA （RACE） and bioinformatics methods were used to isolate CDPKs from common tobacco. A phylogenetic tree was created using the MEGA4.0 program and expression patterns of the three full-length CDPK genes were studied by RT-PCR. After all aforementioned efforts, we obtained eight additional common tobacco CDPK genes, of which three possessed complete open reading frames （ORFs）. Phylogenetic analysis divided 1 1 full-length Nicotiana CDPK genes into four subfamilies, and two putative common tobacco and Arabidopsis orthologous CDPK genes might correspond to well-conserved functions. Three full-length CDPK genes in common tobacco were detected in all tobacco organs tested, but their expression patterns were significantly different. Eight non-redundant common tobacco CDPK genes were isolated in this study. Along with the previously characterized CDPK genes, at least 15 members of the CDPK family exist in common tobacco. This work establishes a foundation for a genome-wide study of this important gene family in common tobacco.

以单细胞压片技术观察烟草(Nicotiana tabacum L.)合子细胞器DNA的分布

通过实验建立了单个合子的压片技术,尝试了将染色与固定分开或同时进行的两种压片方法,两种方法均可以用于不同发育阶段合子细胞器DNA分布的检测,但二者各具特色。同时也利用共聚焦激光扫描显微镜观察了烟草(NicotianatabacumL.)合子细胞器的分布,并与单细胞压片技术进行了比较。实验表明它们各有其优越性。

Nonphytotoxic copper oxide nanoparticles are powerful“nanoweapons”that trigger resistance in tobacco against the soil-borne fungal pathogen Phytophthora nicotianae

Investigations into the potential application of nanoparticles acting as nanofungicides in sustainable agriculture are rapidly expanding due to the high antimicrobial properties of these compounds,which do not risk inducing pathogen resistance to fungicides.A detailed understanding of the impact of copper oxide nanoparticles(CuO NPs)on soil-borne phytopathogenic fungi is yet to be obtained.This study aimed to explore the in vitro antifungal activity and control efficacy of CuO NPs applied via irrigation with respect to tobacco black shank(TBS)disease caused by Phytophthora nicotianae.The results revealed that CuO NPs greatly interfered with the reproductive growth process of this fungus,repressing hyphal growth,spore germination and sporangium production.Additionally,morphological damage,intracellular ROS accumulation and increased SOD enzyme activity in hyphae were the antifungicidal mechanisms of these NPs.In pot experiments,treatment with CuO NPs at 100 mg L^(–1)significantly suppressed TBS development,compared with the effect on control plants,and the control efficacy reached 33.69%without inducing phytotoxicity.Exposure to CuO NPs significantly activated a series of defense enzymes,and resistance genes in tobacco can further explain the mechanisms by which CuO NPs suppressed fungal infection.The Cu content in both the leaves and roots of P.nicotianae-infested plants increased by 50.03 and 27.25%,respectively,after treatment with 100 mg L^(–1)CuO NPs,compared with that of healthy plants.In particular,a higher Cu content was observed in infected roots than in leaves.Therefore,this study showed the potential of CuO NPs applied as nanofungicides and as nanoinducers of fungus resistance genes for the management of TBS through inhibition of pathogen infection and stimulation of plant defenses.

Interference of Quinolinate Phosphoribosyltransferase Gene QPT Affects Agronomic Traits and Leaf Quality in Nicotiana tabacum L.

Quinolinate phosphoribosyltransferase(QPRTase),a key enzyme in ensuring nicotinic acid is available for the synthesis of defensive pyridine alkaloids in Nicotiana species,also plays an important role in nicotinamide adenine dinucleotide(NAD)biosynthesis.In this study,the morphological traits,the quality characteristics and photosynthetic parameters in QPT-overexpressing/interfering tobacco plants were investigated,respectively.Results showed that the interference of QPT gene not only reduced significantly the morphological traits including plant height,stem girth,leaf number and leaf length,etc.at 20 days after transplanting(DAT),but the flowering period was delayed 10-15 d in interfered tobaccos compared with the overexpressed,control and wild-type counterparts.However,at 40 DAT and 60 DAT,only three indexes(plant height,stem girth and leaf number)in QPT-interfering plants appeared significant difference in comparison with other three types of tobacco lines.Meanwhile,the determination results from nicotine,sugar,K+and Cl-content showed the nicotine content in interfered plants was always significantly lower than that in overexpressed plants,control and the wild-type ones respectively whatever toppling or not.At the same time,the toppling treatment also caused the increasement of K+content among the four different tobacco lines,but the maximum increase amplitude of K+content was found in QPT-overexpressing tobaccos while the minimum appeared in QPT-interfering plants.Finally,QPT-interference in transgenic tobaccos likewise affected the photosynthesis by reducing net photosynthetic rate(Pn),stomata conductance(Gs)and transpiration rate(Tr),while there was no significant difference between QPT-overexpressing plants and the controls and the wild-types.

Generation of White Flowers by Transgenic Approach

Flower color is one of the most attractive characteristics in ornamental plants,contributing to the major value in the floricultural market. Anthocyanins are a major colored class of flavonoids that are responsible for the pink,

基于电子鼻技术的烟丝受虫害前后挥发性成分差异分析

针对烟草(Nicotiana tabacum L.)中烟草甲(Lasioderma serricorne)检测技术不完善,检测结果不可靠等问题,以电子鼻检测结果为基础,通过主成分分析法(PCA)和Loading分析法对数据结果进行分析。结果表明,4种烟丝的主要挥发物质为硫类物质、短链烷烃、醇醚醛酮、小分子氮氧化合物等,虫样及性激素的主要挥发性物质是小分子氮氧化合物、硫化物。受虫害后烟丝中硫类化合物明显降低,在对无机硫化物敏感的传感器(W1W)中浓香虫害烟丝的响应值比浓香烟丝下降48.33%,清香虫害烟丝比清香烟丝下降59.48%;在对有机硫化物敏感的传感器(W2W)中浓香虫害烟丝的响应值比浓香烟丝下降35.72%,清香虫害烟丝比清香烟丝下降48.55%。PCA结果显示,4种烟丝样品表现出非常好的区分度,雌虫、雄虫样品在占比高达99.70%的主成分1上几乎没有差别。Loading分析结果显示,烟丝样品中气味主要成分为硫类物质,烟草甲虫样气味主要成分是小分子氮氧化物,其次为硫类物质。

Quality Characteristics of Tobacco Leaves with Different Aromatic Styles from Guizhou Province,China

The relationships between chemical components and quality indexes were studied in the tobacco leaves with different aromatic styles. A total of 16 chemical components, 4 quality indexes, and 6 smoking quality indexes from 366 tobacco leaf samples with 4 different types of aroma from Guizhou Province, China, were subjected to principal component analysis and stepwise regression analysis. The tobacco leaves with different types of aroma showed remarkable difference in the contents of chemical components, quality indexes, and smoking quality indexes. The first principal factors （carbohydrates and nitrogen-containing compounds） of the chemical composition of the leaf were similar among different types of aroma, which showed that the quality of the leaf was mainly influenced by carbohydrates and nitrogen-containing compounds and their ratios. The factors for the second through the seventh principal components varied largely among varioUs aromatic types, suggesting the contribution of other chemical components to the leaf quality. In addition, the smoking quality of four different aromatic leaves showed significant correlation with the different chemical components. The quality of tobacco leaves with different types of aroma was influenced by multiple factors, especially ecological conditions and culture techniques, which may provide guidance for directive cultivation of high-quality tobacco leaves.

Populational Fluctuation and Distribution of Epitrix spp.Foudras(Coleoptera:Chrysomelidae)in the Organic Tobacco Management in Santa Cruz do Sul,RS,Brazil

Among the chrysomelids, some species are considered agricultural pests, such as some representatives of Epitrix spp. Popularly known as tobacco flea, these beetles cause damage to tobacco (Nicotiana tabacum L.) by feeding on the leaf surface, causing injuries, reducing productivity and the final quality of the product to be marketed. However, studies of the biology and interactions of these individuals with the environment become essential in order to develop effective and sustainable control measures. The objective of this study was to analyze the distribution and population fluctuation of Epitrix spp., as well as to evaluate the possible influence of temperature, rainfall and adjacent vegetation on the population dynamics of this beetle in organic tobacco growing in the municipality of Santa Cruz of the South, RS. The collections were carried out weekly during the 2011/2012 harvest, using Malaise and Pit-fall traps, distributed in four points along three lines. A total of 1584 individuals belonging to the genus Epitrix were collected, and their greatest abundance was observed in late January and early February 2012 due to climatic factors, such as rainfall and temperature, favorable to the development of specimens and the tobacco leaf harvesting reduced the availability of food and conditioned a greater movement of these individuals in the crop. The results presented here are of great importance for deciding the best methodology for controlling the smoke flea, as well as knowing the influence of abiotic factors on the behavior of these organisms in the field.

Diversity arrays technology (DArT) for studying the genetic polymorphism of flue-cured tobacco (Nicotiana tabacum)

Diversity arrays technology （DART） is a microarray-based marker system that achieves high throughput by reducing the complexity of the genome. A DArT chip has recently been developed for tobacco. In this study, we genotyped 267 flue-cured cultivars/landraces, including 121 Chinese accessions over five decades from widespread geographic regions in China, 103 from the Americas, and 43 other foreign cultivars, using the newly developed chip. Three hundred and thirty polymorphic DArT makers were selected and used for a phylogenetic analysis, which sug- gested that the 267 accessions could be classified into two subgroups, which could each be further divided into 2-4 sections. Eight elite cultivars, which account for 83% of the area of Chinese tobacco production, were all found in one subgroup. Two high-quality cultivars, HHDJY and Cuibil, were grouped together in one section, while six other high-yield cultivars were grouped into another section. The 330 DArT marker clones were sequenced and close to 95% of them are within non-repetitive regions. Finally, the implications of this study for Chinese flue-cured tobacco breeding and production programs were discussed.

Sesquiterpenes from the leaves of Nicotiana tabacum and their anti-tobacco mosaic virus activity

In searching for more bioactive compounds, phytochemical investigations on the acetone extract of the leaves ofNicotiana tabacurn resulted in the isolation of two new sesquiterpenes, nicosesquiterpene A and B （1 and 2）, along with four known sesquiterpene derivatives （3-6）. Structural elucidation of I and 2 was performed by spectral methods, such as HRMS, IR, UV, 1D and 2D NMR spectroscopy. Compounds 1 and 2 are the first naturally occurring pterosin-type sesquiterpene bearing an isopropyl moiety. Compounds 1-6 were also evaluated for their anti-tobacco mosaic virus （anti-TMV） activity. The results showed that compounds 1 and 2 exhibited high anti-TMV activity with inhibition rates of 36.7% and 45.6%, respectively, which is higher than that of positive control. The other compounds also showed potential activity with inhibition rates in the range of 22.7%-29.2%.

烟草根系形态发育模拟模型

在分析烟草根系生长发育规律的基础上,采用作物模拟技术,定量地描述了根系生长与环境之间的动态关系,建立了烟草根系形态发育的数学模型。根据“根箱法”测定的资料,确定了根系模型的相关参数;通过运行模型,以较少的参数实现了烟草根系数量、长度、根长指数和根长密度等指标的输出,为根系的研究提供了新的手段。利用2003年的试验资料对模型进行检验,结果表明,烟草根系一级、二级侧根根长及其数量、根长密度和根长指数等的模拟值与实测值均呈显著正相关,相关系数分别达0.9540～0.9896,表明模型对烟草根系形态发育动态具有较好的预测效果。

不同有机酸对烤烟品质和产值的影响

采用大田试验研究了苹果酸、油酸和腐殖酸对烤烟品质指标及产值效果的影响。结果表明,3种有机酸能明显提高烟叶中P素、K素、总糖、还原糖含量、钾氯比和糖碱比值,以及糠醛、苯甲醛、茄酮、巨豆三烯酮和新植二烯等主要香气成分的含量,改善烟草的香吃味。同时不同程度地降低烟叶中N素、淀粉、蛋白质、烟碱和氯离子的含量,从而协调和平衡烟叶中的化学成分,改善烟叶的品质。其中,苹果酸的效果最佳,腐殖酸和油酸次之。苹果酸可明显提高烤烟的均价和上中等烟的比例,腐殖酸可明显提高烤烟的产量、产值及上中等烟的比例。

转番茄正义抗坏血酸过氧化物酶基因提高烟草耐盐能力

【目的】探讨叶绿体类囊体膜抗坏血酸过氧化物酶(tAPX)与耐盐性的关系。【方法】从番茄叶片中分离到类囊体膜抗坏血酸过氧化物酶基因(LetAPX),利用农杆菌介导的叶盘法转入到烟草中。Northern杂交分析了LetAPX在盐胁迫(NaCl)下的表达特征。以野生型(WT),转正义LetAPX烟草株系T2-2(+)和T2-6(+)为试材,测定了盐胁迫条件下APX酶活性,过氧化氢(H2O2)含量,种子发芽率,植株的鲜重、干重,光合速率及叶绿素荧光参数。【结果】Northern杂交显示LetAPX已转到烟草基因组中,且基因的表达受盐胁迫的诱导。盐胁迫下转基因烟草的种子发芽率,植株的鲜重与干重,APX酶活性和清除H2O2的能力都显著高于野生型,并且转基因烟草比野生型具有更高的净光合速率(Pn)和PSII最大光化学效率(Fv/Fm)。【结论】LetAPX的过量表达有助于提高烟草的耐盐能力。

我国西南烟区典型植烟土壤烤烟氮素的吸收规律

通采用15N同位素示踪方法,研究我国西南烟区典型红壤、黄壤、水稻土烤烟的氮素吸收规律。结果表明,烤烟氮素的累积与烟叶产量显著正相关,西南烟区烤烟氮适宜需求量为60～100kghm-2。不同土壤类型上种植的烤烟,其氮吸收差异显著,云南红壤烤烟生长前期的氮素吸收速率最高,其次是水稻土烤烟,黄壤烤烟最低;相应氮素吸收高峰分别在移栽后7、9和11周。烤烟中总氮、土壤氮、肥料氮的吸收速率均呈单峰曲线变化,且肥料氮的吸收高峰早于土壤氮;烤烟进入旺长期以后,土壤氮的吸收速率逐渐高于肥料氮,转入以吸收土壤氮为主。西南烟区烤烟打顶前土壤氮的累积比例为59.8%,肥料氮为72.1%,不同土壤类型烤烟打顶前氮素累积比例差异显著,红壤、黄壤、水稻土烤烟分别为87.8%、47.3%和49.2%。因此,根据不同土壤类型烤烟氮素吸收动态,在适宜的氮素需求量下,应以烤烟打顶前氮素需求量和土壤氮素供应量来计算烤烟氮肥需求量。

光质对烟草叶片生长发育过程中抗氧化系统的影响

以云烟87植株为材料,通过覆盖白、红、黄、蓝、紫色滤膜获得不同光质,于大田条件下研究了光质对烟草叶片生长发育过程中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）、抗坏血酸过氧化物酶（APX）、谷胱甘肽过氧化物酶（GPX）、谷胱甘肽还原酶（GR）等抗氧化酶活性,抗氧化剂谷胱甘肽（GSH）和抗坏血酸（AsA）以及丙二醛（MDA）含量的影响.结果表明,在烟草植株第11片叶生长发育的7-70 d内,其抗氧化酶活性和抗氧化物质含量呈现先升高后下降的变化趋势.与白光（对照）相比,黄光诱导烟草叶片SOD、CAT、APX和GR活性升高,以及AsA和GSH含量增加;而红光诱导APX和GR活性上升,以及GSH和AsA含量升高;但紫光却使SOD、CAT、POD、GR和GPX活性下降,GSH和AsA含量降低,而蓝光则使所有抗氧化酶活性和抗氧化物质含量降低.紫光和蓝光处理的烟草叶片中MDA含量较高,而黄光和红光处理的则较低.总体而言,在大田条件下,相对红光和黄光而言,蓝光和紫光处理下的烟草叶片更容易发生光氧化胁迫.

施用纳米碳对烤烟氮素吸收和利用的影响

为明确纳米碳在提高烤烟氮素吸收利用方面的效果,在盆栽条件下,研究了纳米碳不同用量对烤烟根系生长发育、干物质积累和氮素吸收利用的影响。结果表明,在常规肥料中添加纳米碳能够促进烤烟根系生长发育,明显提高烟株根系活力和单株根系生物量,增加植株干物质积累量。施用纳米碳增加了烤烟植株成熟期各器官氮素含量和积累量,而未明显影响氮素在植株不同器官的分配。施用纳米碳不仅增加了植株对肥料氮的吸收量,还增加了对土壤氮的吸收量,这与其促进烤烟根系生长发育、提高根系吸收能力有密切关系。纳米碳无论做基肥还是做追肥,均显著提高了氮肥利用率,提高幅度分别达到14.44%和9.62%,有效降低了氮素土壤残留和损失。

机械损伤对烤烟植株氮素吸收及体内烟碱含量的影响

采用砂培和营养液培养方法,研究了在营养生长阶段切除顶芽和叶片损伤对烟株生长、体内烟碱浓度、氮浓度及吸氮量的影响。结果表明,切除顶芽导致烟株体内烟碱浓度和含量显著增加。叶片损伤也能增加体内的烟碱浓度,且损伤二次比损伤一次的效果更为明显,说明叶面损伤对烟株体内烟碱合成的影响具有累积效应。但叶面损伤对烟株体内烟碱合成的影响小于打顶处理。切除顶芽和叶面损伤对烟株体内的氮浓度及吸氮量均无显著影响,但显著提高了烟株体内单位氮素产生烟碱的能力。试验结果还表明,机械损伤刺激烟株体内烟碱的合成,烟碱增加与氮素吸收无直接关系。

氮磷钾肥及水分因子对烤烟叶片叶黄素的综合影响

通过盆栽试验探讨了氮、磷、钾肥及水分因子对烤烟叶片叶黄素的综合影响。交互效应测验表明,影响烤后烟叶叶黄素含量的一级(2因素)和三级(4因素)交互作用显著,二级(3因素)交互作用中以氮磷水、磷钾水的交互作用为主。一级交互效应分析表明,氮钾、氮磷、磷钾、磷水、钾水互作对烤后烟叶叶黄素含量的影响表现协同促进作用。氮水间互作中存在一个阈值,低于这个阈值时氮水间互作对烤后烟叶叶黄素含量的影响表现协同促进作用,高于这个阈值时表现拮抗作用。二级、三级交互效应分析表明,氮磷钾合理配施(1∶1.3∶3.1)和适量浇水明显促进叶黄素的积累。当施氮量较少时增施磷肥和适量浇水,当施磷量较少时增施氮钾肥和适量浇水,以及当土壤相对含水量较低时增施氮钾肥均可以提高烤烟叶片叶黄素含量。结果表明,高氮肥(每盆施氮量大于6g纯氮)、高钾肥(每盆施钾量大于18gK2O)、中等磷肥配合适量浇水(74.3%～79.5%的土壤相对含水量)明显促进叶黄素的积累。